Methods and materials relating to novel CD39-like polypeptides

ABSTRACT

The invention provides polynucleotides isolated from cDNA libraries of human fetal liver-spleen and macrophage as well as polypeptides encoded by these polynucleotides and mutants or variants thereof. The polypeptides correspond to a human CD39-like protein. Other aspects of the invention include vectors containing polynucleotides of the invention and related host cells as well a processes for producing CD39-like polypeptides, and antibodies specific for such polypeptides.

1. RELATED APPLICATIONS

This patent application is a continuation-in-part of PCT patentapplication Ser. No. PCT/US99/16180 filed Jul. 16, 1999 which is acontinuation-in-part of U.S. patent application Ser. No. 09/350,836filed Jul. 9, 1999 now U.S. Pat. No. 6,387,645 which is acontinuatiqn-in-part of U.S. patent application Ser. No. 09/273,447filed Mar. 19, 1999, now abandoned the disclosures of all of which areincorporated by reference herein in their entirety. The disclosures ofco-owned, co-pending U.S. patent application Ser. No. 09/122,449 filedJul. 24, 1998, and U.S. patent application Ser. No. 09/118,205 filedJul. 16, 1998, are incorporated by reference herein in their entirety.

2. FIELD OF THE INVENTION

This invention relates in general to novel polynucleotides isolated fromcDNA libraries of human fetal liver-spleen and macrophages and topolypeptides encoded by these polynucleotides. In particular, theinvention relates to a human CD39-like protein with homologies to ATPdiphosphohydrolases and variants thereof.

3. BACKGROUND

CD39 (cluster of differentiation 39) is a cell-surface moleculerecognized by a “cluster” of monoclonal antibodies that can be used toidentify the lineage or stage of differentiation of lymphocytes and thusto distinguish one class of lymphocytes from another. This CD39 moleculewas originally defined as a B lymphocyte marker (Rowe, M., et al. Int.J. Cancer 29:373 (1982)). Subsequent studies have shown CD39 to be amarker for a distinct subset of activated lymphocytes within theallosensitized CD8-positive cytotoxic cells (Gouttefangeas C., et al.,Eur. J. Immunol. 22:2681 (1992)). Outside of lymphoid tissue, CD39 canbe found in quiescent vascular endothelial cells (Kansas, G. S., et al.,J. Immunol. 146:2235 (1991)) and throughout rat brain in the neurons ofthe cerebral cortex, hippocampus, and cerebellum, as well as in glialcells (Wang, T-F. and Guidotti, G., Brain Res. 790:318 (1998)).

CD39 is a 510-amino acid protein with a predicted molecular mass of 57kDa. However, because of heavy glycosylation at asparagine residues (sixpotential N-glycosylation sites) the molecule displays a mobility closerto 100 kDa (Maliszewski, C. R., et al., J. Immunol. 153:3574 (1994)).CD39 contains two hydrophobic regions, one near the amino terminus andthe other near the carboxyl terminus which are believed to betransmembrane regions.

The role of CD39 in platelet aggregation and ATP/ADP hydrolysis isunclear. Although CD39 was originally reported to be an ectoADPase witha preference for ATP over ADP as a substrate, Wang, et al., J. Biol.Chem. 271:9898-9901(1996), Marcus, et al., J. Clin. Invest. 99:1351-1360(1997) reported that CD39 was unique for its high preference for ADPover ATP as a substrate and in 1998, Gayle, et al., J. Clin Invest.10:1851-1859 (1998), described CD39 as an ectoADPase with no preferencefor one substrate over the other.

Reports that several ATP Diphosphohydrolases (ATPDases) share amino acidsequence homology with CD39 have been substantiated by the showing thatCD39 is itself an ATPDase (Wang, T-F., et al., J. Biol. Chem. 271:9898(1996); Kaczmarek, E., et al., J. Biol. Chem. 271:33116 (1996)). SinceCD39 is a plasma membrane-bound enzyme, CD39 has been termed an“ecto-ATPase,” but CD39 is more often referred to as an “ecto-apyrase”because of the reduced rate of hydrolysis of ADP when compared withecto-ATPases.

This activity has shown to modulate platelet reactivity and aggregationin response to vascular injury. During vascular injury, activatedplatelets aggregate forming an occlusive thrombus. Excessive plateletaccumulation at sites of vascular injury can contribute to vesselocclusion. Endothelial cells respond to the potentially occlusiveeffects of platelet aggregation by several mechanisms. One of thesemechanisms results ecto-apyrase-mediated removal of ADP, which in turneliminates platelet reactivity and recruitment. It is now known that theendothelial ecto-apyrase responsible for this ADP removal is CD39(Marcus, A. J., et al., J. Clin. Invest. 99:1351 (1997)).

Recently, CD39 was engineered to produce a soluble form of the molecule.This soluble CD39 was shown to display the same nucleotidase activity asthe membrane-bound molecule (Gayle, R. B., et al., J. Clin. Invest.101:1851 (1998)). Intravenously administered soluble CD39 also remainedactive in mice for an extensive period of time, indicating that solubleCD39 could be useful as a inhibitor of platelet aggregation in theprophylaxis or treatment of platelet-mediated thrombotic conditions.

Platelet aggregation inhibitors (antithrombotic agents) decrease theformation or the action of chemical signals that promote plateletaggregation. Currently available antithrombotic agents include aspirin,ticlopidine, and dipyridamole. These agents have proven beneficial inthe prevention and treatment of occlusive cardiovascular diseases,including myocardial infarction, cerebral ischemia, angina.Antithrombotic therapy has also been used in the maintenance of vasculargrafts.

Myocardial infarction is the development of necrosis of the myocardium(the middle muscular layer of the heart wall) due to a criticalimbalance between oxygen and myocardial demand. The most common cause ofacute myocardium infarction is narrowing of the epicardial blood vesselsdue to atheromatous plaques. Plaque rupture with subsequent exposure ofbasement membrane results in platelet aggregation and thrombusformation, which can result in partial or complete occlusion of thevessel and subsequent myocardial ischemia.

In cerebral ischemia, inadequate blood flow results from an occlusion ina blood vessel or hemorrhaging. In the latter case, excessive bleedingin one area of the brain deprives another area of blood. If the damageoccurs in a singular small area, “transient” or “focused” cerebralischemia results. When a major artery is blocked (carotid artery) globalor diffused ischemia results. The primary medical strategy for secondaryprevention of stroke is antiplatelet therapy. Aspirin is currentlyemployed for reducing the risk of recurrent transient ischemic attacksor stroke in men who have transient ischemia of the brain due to fibrinemboli.

Each year, thousands of patients suffer a decline in blood flow to oneor more limbs. Without sufficient blood flow, and, unless blood flow canbe restored in time, the limb must be amputated. In some cases, graftsfrom the patient's veins can be used to form new arteries. However, incases where the quality of the veins is poor, polymeric vascular graftsare typically used. The polymeric grafts are inherently thrombogenic asthe blood constituents passing through the grafts become activated andtend to form clots. Efforts to line the grafts with endothelial cellscan reduce blood clotting, but better results are obtained whenantithrombotic therapy is employed.

Angina pectoris is a characteristic chest pain caused by inadequateblood flow through the blood vessels of the myocardium. The imbalancebetween oxygen delivery and utilization may result from a spasm of thevascular smooth muscle or from obstruction of blood vessels caused byatherosclerotic lesions. Three classes of drugs have been shown to beeffective in treating angina: nitrates, beta-blockers and calciumchannel blockers. Currently, the antithrombotics dipyridamole andaspirin are employed to prophylactically treat angina pectoris.

Ecto-apyrases, such as CD39, offer a number of advantages over severalof the standard antithrombotics. For example, aspirin treatment controlsthe prothrombotic action of thromboxane; however, aspirin also preventsformation of antithrombotic prostacyclin, which limits aspirin'sefficacy. Another antithrombotic, endothelium-derived relaxing factor(nitric oxide; “EDRF/NO”), is inhibited in vitro and in vivo byhemoglobin after its rapid diffusion into erythrocytes. In contrast,CD39 is aspirin-insensitive and completely inhibits platelet reactivityeven when eicosanoid and EDRF/NO production are blocked.

CD39's ATPDase activity also implicates CD39 in the modulation ofneurotransmission. ATP is a major purinergic neurotransmitter that isoften co-released into the synaptic cleft with severalneurotransmifters. Responses to ATP are mediated by specific plasmamembrane receptors, called P2 purinergic receptors (Dubyak, G. R. andEl-Motassim,C. Am J. Physiol. 34:C577-C606 (1993)). The distribution ofCD39 in the rat brain indicates that CD39 plays a role in terminating P2purinergic neurotransmission (Wang, T. F. and Guidotti, G., Brain Res.790:318 (1998)). Furthermore, a decrease in ecto-apyrase activity isbelieved to lead to an accumulation of the excitatory neurotransmitter,extracellular ATP, as well as a deficiency of the endogenousanticonvulsant extracellular adenosine.

The chomosomal localization of CD39 provides additional support for arole in modulation of neurotransmission. More specifically, CD39 hasbeen mapped to chromosome 10q 23.1-24.1 (Maliszewski, C. R., et al., J.Immunol. 153:3574 (1994)), and this site overlaps with thesusceptibility locus for human partial epilepsy with audiogenic symptoms(Ottman, R. et al., Nature Genet. 10:56 (1995)). This co-localization ofthe CD39 gene and the susceptibility locus has led to the hypothesisthat decrease in ecto-apyrase activity in the brain is the primary causeof partial epilepsy (Wang T-F., et al., Mol. Brain Res. 47:295 (1997)).

A screen for human cDNAs that hybridize to cosmids from the humanchromosome 9q34 region lead to the identification of a transcript withhigh homology to a chicken muscle ecto-ATPase (60% identity) and theecto-apyrase CD39 (41% amino acid identity) (Chadwick, B. P., Mamm.Genome 8:668 (1997)). This gene, designated “CD39-like-1 gene” (CD39L1),has a higher degree of homology to CD39 than does chicken muscleecto-ATPase. The biological activity of this protein has not been testedbut on the basis of the high amino acid homology, CD39L1 is believed tobe a new member of the ecto-ATPase family. Recently, a mouse gene withhomology to NTPases was cloned and sequenced (Acc. No. AF006482) byChadwick et al. (Mamm. Gen. 9:162-164 (1998).)

4. SUMMARY OF THE INVENTION

The invention is based on polynucleotides isolated from cDNA librariesprepared from human fetal liver-spleen and macrophages. The compositionsof the present invention include novel isolated polypeptides withapyrase and/or NDPase activity, in particular, novel human CD39-likepolypeptides, and active variants thereof, isolated polynucleotidesencoding such polypeptides, including recombinant DNA molecules, clonedgenes or degenerate variants thereof, especially naturally occurringvariants such as allelic variants, antisense polynucleotide molecules,and antibodies that specifically recognize one or more epitopes presenton such polypeptides, as well as hybridomas producing such antibodies.

The compositions of the invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

The isolated polynucleotides of the invention include naturallyoccurring or wholly or partially synthetic DNA, e.g., cDNA and genomicDNA, and RNA, e.g., mRNA. One polynucleotide according to the inventionencodes a novel CD39-like protein having the amino acid sequence shownin FIG. 2 (SEQ ID NO. 3), which has been designated CD39-L4. Anotherpolynucleotide according to the invention encodes a novel CD39-likeprotein having the amino acid sequence shown in SEQ ID NO: 27, which hasbeen designated CD39-L2. In another embodiment, a polynucleotideaccording to the invention encodes a novel CD39-like protein having thefull length or mature amino acid sequence set forth in SEQ ID NO. 25,which has been designated CD39-L66, and is an isoform of CD39-L4. Theisolated polynucleotides of the invention include a polynucleotidecomprising the nucleotide sequence of SEQ ID NO. 2, 24 or 26. Thepolynucleotides of the invention also include polynucleotides thatencode polypeptides with a biological activity of the polypeptide of SEQID NO. 3 or 27 (including apyrase or NDPase activity) such as (a) thenucleotide sequence of SEQ ID NO. 2, 24, 26 or (b) a nucleotide sequenceencoding the full length or mature amino acid sequence of SEQ ID NO. 3,25, or 27; (c) a polynucleotide which is an allelic variant of anypolynucleotide recited above; (d) a polynucleotide that hybridizes understringent conditions to (a) or (b); (e) or a polynucleotide that encodesa polypeptide comprising at least one CD39-like domain, e.g. catalyticdomain.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

The invention also provides a polynucleotide including a nucleotidesequence that is substantially equivalent to these polynucleotides.Polynucleotides according to the invention can have at least about 80%,more typically at least about 90%, and even more typically at leastabout 95%, sequence identity to a polynucleotide of SEQ ID NO. 2, 24 or26 and specifically include a human polynucleotide which has at least80% sequence identity to a polynucleotide of SEQ ID NO. 2, 24 or 26; ora polynucleotide which has at least 90% sequence identity to apolynucleotide of SEQ ID NO. 2, 24 or 26. Similarly, polypeptides of theinvention include polypeptides having apyrase or NDPase activity and atleast about 80%, 90% or 95% sequence identity to SEQ ID NO. 3, 25 or 27.

A further aspect of the invention is the development of novel CD39-L4variants which preferably have improved ADPase or NDPase activitycompared to wild type CD39-L4 (SEQ ID NO: 5). This aspect of theinvention includes polypeptides comprising at least one amino acidsubstitution selected from the group consisting of: D168→T, S170→Q andL175→F, wherein said substitution(s) result in increased ADPase activityof the polypeptide. One preferred embodiment is the polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 7 (encoded by thenucleotide sequence of SEQ ID NO. 6), which is a variant CD39-L4containing all three substitutions that has been designated ACRIII. Aplasmid containing this DNA was deposited with the American Type CultureCollection (ATCC), 10801 University Avenue, Manassas, Va., on Jul. 13,1999 under the terms of the Budapest Treaty (ATCC accession numberPTA-346). Alternatively, instead of making the specific D168→T, S170→Qand/or L175→F substitution(s), substitution of amino acids with similarproperties is contemplated. Additional conservative substitutions atamino acid positions other than D168, S170 and/or L175 are furthercontemplated. For example, all of the corresponding amino acids fromCD39 could be substituted for amino acids 167-181 of CD39-L66 orCD39-L4.

In addition, development of novel CD39-L2 variants which preferably haveimproved ADPase or NDPase activity compared to wild type CD39-L2 (SEQ IDNO: 27) is also contemplated. This aspect of the invention includespolypeptides comprising at least one amino acid substitution whereinsaid substitution(s) result in increased ADPase activity of thepolypeptide.

Polynucleotides encoding these polypeptides, vectors and host cellscomprising such polynucleotides, methods of using such host cells toproduce polypeptides, and other therapeutic products comprising thepolypeptides (including fusion proteins in which the CD39-likepolypeptide is fused to a heterologous peptide or polypeptide, such asan immunoglobulin constant region, or derivatives in which the CD39-likepolypeptide is modified by water soluble polymers to increase itshalf-life) are also comprehended by the invention, as are methods oftreating a subject suffering from a disorder relating to thrombosis,coagulation or platelet aggregation by administering such therapeuticproducts.

The invention further comprises methods of inhibiting plateletaggregation in a mammalian subject by reducing the ratio of ADP:ATP in amammalian subject to a less than normal ratio by administering thepolypeptides of the invention or by administering polypeptides withADPase activty and at least about 90% sequence identity to SEQ ID NO: 3,25 or 27. Preferably the ratio of ADP:ATP is reduced withoutsignificantly affecting ATP levels. In one embodiment, the ADP:ATP ratiois reduced systemically in circulation. In another embodiment, theADP:ATP ratio is reduced locally, for example, in heart, brain, kidney,lungs, limbs or other organs.

Methods of identifying compounds capable of reducing the ratio ofADP:ATP to a less than normal ratio are also contemplated. For example,compounds may be identified by steps including: determining apyraseactivity of said compound on ATP; determining apyrase activity of saidcompounds on ADP; and selecting a compound that has greater activitywith respect to ADP compared to ATP. Exemplary compounds to be screenedinclude, but are not limited to, CD39-L4 and CD39-L2 variants.

Gene therapy techniques are also provided to modulate disease statesassociated with CD39-L4 or CD39-L2 expression and/or biologicalactivity. Delivery of a functional CD39-L4 or CD39-L2 gene toappropriate cells is effected ex vivo, in situ, or in vivo by use ofvectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or ex vivo by use of physicalDNA transfer methods (e.g., liposomes or chemical treatments).

The invention also relates to methods for producing polypeptides of theinvention comprising growing a culture of cells of the invention in asuitable culture medium under conditions permitting expression of thedesired polypeptide, and purifying the protein from the cells or theculture medium. Preferred embodiments include those in which the proteinproduced by such process is a mature form of the protein.

Protein compositions of the present invention, including therapeuticcompositions, comprise polypeptides of the invention and optionally anacceptable carrier, such as a hydrophilic (e.g., pharmaceuticallyacceptable) carrier.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asoligomers for PCR, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. For example, becausethe expression of CD39-L4 and CD39-L2 mRNA is largely restricted tospecific tissues (CD39-L4 in macrophages and CD39-L2 in adult heart andfetal brain), polynucleotides of the invention can be used ashybridization probes to detect the presence of specific mRNA in a sampleusing, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath, et al.,Science 258:52-59 (1992), as expressed sequence tags for physicalmapping of the human genome.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook, J., et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, a polypeptide of the invention can be used togenerate an antibody which specifically binds the polypeptide. Thepolypeptides of the invention having ATPDase activity are also usefulfor inhibiting platelet aggregation and can therefore be employed in theprophylaxis or treatment of pathological conditions caused by theinflammatory response. The polypeptides of the invention can also beused as molecular weight markers, and as a food supplement.

Another aspect of the invention is an antibody that specifically bindsthe polypeptide of the invention. Such antibodies can be eithermonoclonal or polyclonal antibodies, as well fragments thereof andhumanized forms or fully human forms, such as those produced intransgenic animals. The invention further provides a hybridoma thatproduces an antibody according to the invention and anti-idiotypeantibodies.

Antibodies of the invention are useful for detection and/or purificationof the polypeptides of the invention.

Methods are also provided for preventing, treating or ameliorating amedical condition, including thrombotic diseases, which comprisesadministering to a mammalian subject, including but not limited tohumans, a therapeutically effective amount of a composition comprising apolypeptide of the invention or a therapeutically effective amount of acomposition comprising a binding partner of (e.g., antibody specificallyreactive for) CD39-like polypeptides of the invention. The mechanics ofthe particular condition or pathology will dictate whether thepolypeptides of the invention or binding partners (or inhibitors) ofthese would be beneficial to the individual in need of treatment.

The invention also provides a method of inhibiting platelet functioncomprising administering a CD39-L4 or CD39-L2 polypeptide of theinvention to a medium comprising platelets. According to this method,polypeptides of the invention can be administered to produce an in vitroor in vivo inhibition of platelet function. A polypeptide of theinvention can be administered in vivo as antithrombotic agent alone oras an adjunct to other therapies.

Also provided are methods of hydrolyzing nucleotidediphosphatescomprising administering CD39-L4 or CD39-L2 polypeptides of theinvention to a medium comprising nucleotidediphosphates. According tothis method, polypeptides of the invention can be administered toproduce an in vitro or in vivo hydrolysis of nucleotidediphosphates. Apolypeptide of the invention can be administered in vivo alone or as anadjunct to other therapies.

The invention further provides methods for manufacturing medicamentsuseful in the above described methods relating to platelet aggregationand thrombosis.

The invention also provides methods for detecting or quantitating thepresence of the polynucleotides or polypeptides of the invention in atissue or fluid sample, and corresponding kits that comprise suitablepolynucleotide probes or antibodies, together with an optionalquantitative standard. Such methods and kits can be utilized as part ofprognostic and diagnostic evaluation of patients and for theidentification of subjects exhibiting a predisposition to plateletmediated conditions.

The invention also provides methods for the identification of compoundsthat modulate (i.e. increase or decrease) the expression or activity ofthe polynucleotides and/or polypeptides of the invention. Such methodscan be utilized, for example, for the identification of compounds andother substances that interact with (e.g., bind to) the polypeptides ofthe invention, and assays for identifying compounds and other substancesthat enhance or inhibit the activity of the polypeptides of theinvention, such assays comprising the step of measuring activity of suchpolypeptides in the presence and absence of the test compound.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows polynucleotide sequences according to the invention. SEQ IDNO:1 was obtained from the b2HFLS20W cDNA library using standard PCR,sequencing by hybridization signature analysis, and single pass gelsequencing technology. A-adenosine; C-cytosine; G-guanosine; T-thymine.Ambiguous positions are designated as follows: R indicates A or G; Mindicates A or C; W indicates A or T; Y indicates C or T; S indicates Cor G; K indicates G or T; V indicates A or C or G; H indicates A or C orT; D indicates A or G or T; B indicates C or G or T; and N -indicatesany of the four bases.

SEQ ID NO:2 is an extended version of SEQ ID NO:1 which was obtained asdescribed in Example 2.

FIG. 2 shows an amino acid sequence corresponding to the polynucleotidesequence of SEQ ID NO:2. This sequence is designated as SEQ ID NO:3. Theopen reading frame encoding SEQ ID NO:3 begins at nucleotide 246(numbered from the 5′ end) of SEQ ID NO:2. A-Alanine; R-Arginine;N-Asparagine; D-Aspartic Acid; C-Cysteine; E-Glutamic Acid; Q-Glutamine;G-Glycine; H-Histidine; I-Isoleucine; L-Leucine; K-Lysine; M-Methionine;F-Phenylalanine; P-Proline; S-Serine; T-Threonine; W-Tryptophan;Y-Tyrosine; V-Valine; X-any of the twenty amino acids.

FIGS. 3A and 3B show the amino acid sequence alignment of SEQ ID NO:3(identified as “246 prot”) and human CD39 (“CD39Human.seq”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedusing the J. Hein method with the PAM250 residue weight table. Gaps areindicated by dashes; residues that are identical between the twosequences (within 1 distance unit) are boxed.

FIGS. 4A and 4B show the amino acid sequence alignment of SEQ ID NO:3(identified as “264 prot”) and murine NTPase (“mur ntpase”). The aminoacid residues are designated as for FIG. 2. The alignment was generatedas discussed for FIGS. 3A and 3B. Gaps are indicated by dashes; residuesthat are identical between the two sequences (within 1 distance unit)are boxed.

FIG. 5 shows the apyrase conserved regions (ACR) in CD39-L4 in bold. ACRI starts at Phe 53, ACR II starts at Pro 124 and ACR III starts at Met167. The boxed sections highlight the amino acid substitutions that weremade in the wild type CD39-L4 amino acid sequence to form mutantsdesignated ACRI, ACRII and ACRIII.

FIG. 6 (SEQ ID NOS: 6 and 7) shows the nucleotide and correspondingamino acid sequences of a preferred ACRIII mutant containing thefollowing substitutions in the wild type CD39-L4 amino acid sequence:D168→T, S170→Q and L175→F. Changes in both sequences are shown in boldand are underlined. The G to A and A to C changes at positions 502 and503 produce a Thr, the T to C, C to A and C to A changes at positions508-510 result in a Gln and the A to C changed at position 525 resultsin a Phe.

FIG. 7 shows the ADPase activity of CD39-L4 variants ACRI, ACRII andACRIII in comparison to wild type CD39-L4: (1) CD39-L4 ACR I mutant; (2)CD39-L4 ACR II mutant; (3) CD39-L4 ACR III mutant; (4) CD39-L4 wildtype; (5) sCD39; and (6) pSecTag2 vector (Invitrogen).

FIG. 8 shows the amino acid sequence alignment of SEQ ID NO. 3, SEQ IDNO. 25 (previously identified as SEQ ID NO. 5 in FIG. 5 of U.S. Ser. No.09/122,449) and human CD39 (“CD39Human.seq”) (SEQ ID NO: 38). Thealignment was generated using the Jotun Hein method with the PAM250residue weight table. Gaps are indicated by dashes; residues that areidentical between the two sequences (within 1 distance unit) are boxed.

FIG. 9 shows the amino acid sequence alignment of SEQ ID NO. 3, SEQ IDNO. 25 (previously identified as SEQ ID NO. 5 in FIG. 6 of U.S. Ser. No.09/122,449) and the murine NTPase (“mur ntpase”) (SEQ ID NO: 39). Thealignment was generated as discussed for FIG. 8. Gaps are indicated bydashes; residues that are identical between the two sequences (within 1distance unit) are boxed.

6. DETAILED DESCRIPTION 6.1 Definitions

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid” and“polynucleotide” are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic nucleic acid which is capable of being expressed ina recombinant transcriptional unit comprising regulatory elementsderived from a microbial or viral operon.

An “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,”or “segment” is a stretch of polypeptide nucleotide residues which islong enough to use in polymerase chain reaction (PCR) or varioushybridization procedures to identify or amplify identical or relatedparts of mRNA or DNA molecules. “Oligonucleotides” or “nucleic acidprobes” are prepared based on the cDNA sequence provided in the presentinvention. Oligonucleotides comprise portions of the DNA sequence havingat least about 15 nucleotides and usually at least about 20 nucleotides.Nucleic acid probes comprise portions of the sequence having fewernucleotides than about 6 kb, usually fewer than about 1 kb. Afterappropriate testing to eliminate false positives, these probes may beused to determine whether mRNAs are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh, P. S., et al (1992 PCR Methods Appl 1:241-250).

The term “probes” includes naturally occurring or recombinant single- ordouble-stranded nucleic acids or chemically synthesized nucleic acids.They may be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J., et al(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M., et al (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both incorporatedherein by reference.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. An exemplary set of conditionsinclude a temperature of 60-70° C., (preferably about 65° C.) and a saltconcentration of 0.70 M to 0.80 M (preferably about 0.75M). Furtherexemplary conditions include, hybridizing conditions that (1) employ lowionic strength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

The term “recombinant,” as used herein, means that a polypeptide orprotein is derived from recombinant (e.g., microbial or mammalian)expression systems. “Microbial” refers to recombinant polypeptides orproteins made in bacterial or fungal (e.g., yeast) expression systems.As a product, “recombinant microbial” defines a polypeptide or proteinessentially free of native endogenous substances and unaccompanied byassociated native glycosylation. Polypeptides or proteins expressed inmost bacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern different from that expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. The expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an N-terminal methionineresidue. This residue may or may not be subsequently cleaved from theexpressed recombinant protein to provide a final product.

“Recombinant expression system” means host cells which have stablyintegrated a recombinant transcriptional unit into chromosomal DNA orcarry the recombinant transcriptional unit extrachromosomally. The cellscan be prokaryotic or eukaryotic. Recombinant expression systems asdefined herein will express heterologous polypeptides or proteins upon.induction of the regulatory elements linked to the DNA segment orsynthetic gene to be expressed.

The term “open reading frame,” ORF, means a series of triplets codingfor amino acids without any termination codons and is a sequencetranslatable into protein.

The term “expression modulating fragment,” EMF, means a series ofnucleotide molecules which modulates the expression of an operablylinked ORF or EMF. As used herein, a sequence is said to “modulate theexpression of an operably linked sequence” when the expression of thesequence is altered by the presence of the EMF. EMFs include, but arenot limited to, promoters, and promoter modulating sequences (inducibleelements). One class of EMFs are fragments which induce the expressionof an operably linked ORF in response to a specific regulatory factor orphysiological event.

As used herein, an “uptake modulating fragment,” UMF, means a series ofnucleotide molecules which mediate the uptake of a linked DNA fragmentinto a cell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems known in theart.

The presence and activity of a UMF can be confirmed by attaching thesuspected UMF to a marker sequence. The resulting nucleic acid moleculeis then incubated with an appropriate host under appropriate conditionsand the uptake of the marker sequence is determined. As described above,a UMF will increase the frequency of uptake of a linked marker sequence.

“Active” refers to those forms of the polypeptide which retain thebiologic and/or immunologic activities of any naturally occurringpolypeptide.

“Naturally occurring polypeptide” refers to polypeptides produced bycells that have not been genetically engineered and specificallycontemplates various polypeptides arising from post-translationalmodifications of the polypeptide including, but not limited to,acetylation, carboxylation, glycosylation, phosphorylation, ipidationand acylation.

“Derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

“Recombinant variant” refers to any polypeptide differing from naturallyoccurring polypeptides by amino acid insertions, deletions, andsubstitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine, i.e., conservative amino acid replacements. “Insertions” or“deletions” are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a mutant sequence varies from one of those listed hereinby no more than about 20% (i.e., the number of substitutions, additions,and/or deletions in a mutant sequence, as compared to the correspondinglisted sequence, divided by the total number of residues in the mutantsequence is about 0.2 or less). Such a mutant sequence is said to have80% sequence identity to the listed sequence. In one embodiment, amutant sequence of the invention varies from a listed sequence by nomore than 10% (90% sequence identity), in a variation of thisembodiment, by no more than 5% (95% sequence identity), and in a furthervariation of this embodiment, by no more than 2% (98% sequenceidentity). Mutant amino acid sequences according to the inventiongenerally have at least 95% sequence identity with a listed amino acidsequence, whereas mutant nucleotide sequence of the invention can havelower percent sequence identities. For the purposes of the presentinvention, sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence should be disregarded.

Where desired, an expression vector may be designed to contain a “signalor leader sequence” which will direct the polypeptide through themembrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polypeptide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

“Activated” cells as used in this application are those which areengaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed.

The term “intermediate fragment” means a nucleic acid between 5 and 1000bases in length, and preferably between 10 and 40 bp in length.

Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

6.2 Hybridization Conditions

Suitable hybridization conditions may be routinely determined byoptimization procedures or pilot studies. Such procedures and studiesare routinely conducted by those skilled in the art to establishprotocols for use in a laboratory. See e.g., Ausubel, et al., CurrentProtocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989);Sambrook, et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols.1-3, Cold Springs Harbor Press (1989); and Maniatis, et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold SpringHarbor, N.Y. (1982), all of which are incorporated by reference herein.For example, conditions such as temperature, concentration ofcomponents, hybridization and washing times, buffer components, andtheir pH and ionic strength may be varied.

6.3 Nucleic Acids of the Invention

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof. Allelic variations can be routinelydetermined by comparing the sequence provided in SEQ ID NOs: 1, 2, 24 or26, a representative fragment thereof, or a nucleotide sequence at least99.9% identical to SEQ ID NO: 1, 2, 24 or 26 with a sequence fromanother isolate of the same species. Furthermore, to accommodate codonvariability, the invention includes nucleic acid molecules coding forthe same amino acid sequences as do the specific ORFs disclosed herein.In other words, in the coding region of an ORF, substitution of onecodon for another which encodes the same amino acid is expresslycontemplated.

Any specific sequence disclosed herein can be readily screened forerrors by resequencing a particular fragment, such as an ORF, in bothdirections (i.e., sequence both strands).

The present invention further provides recombinant constructs comprisinga nucleic acid having the sequence of any one of SEQ ID NO: 1, 2, 24 or26, the mature protein coding sequence or a fragment thereof. Therecombinant constructs of the present invention comprise a vector, suchas a plasmid or viral vector, into which a nucleic acid having thesequence of any one of SEQ ID NO: 1, 2, 24 or 26 or a fragment thereofis inserted, in a forward or reverse orientation. In the case of avector comprising one of the ORFs of the present invention, the vectormay further comprise regulatory sequences, including for example, apromoter, operably linked to the ORF. For vectors comprising the EMFsand UMFs of the present invention, the vector may further comprise amarker sequence or heterologous ORF operably linked to the EMF or UMF.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available for generating therecombinant constructs of the present invention. The following vectorsare provided by way of example. Bacterial: pBs, phagescript, PsiX174,pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, PMSG, pSVL(Pharmacia).

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced or derepressed by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequences that hybridize under stringent conditions toa fragment of the DNA sequences in FIG. 1, which fragment is greaterthan about 10 bp, preferably 20-50 bp, and even greater than 100 bp,including 200 bp or greater, 300 bp or greater, 400 bp or greater, and500 bp or greater.

In accordance with the invention, polynucleotide sequences which encodethe novel nucleic acids, or functional equivalents thereof, may be usedto generate recombinant DNA molecules that direct the expression of thatnucleic acid, or a functional equivalent thereof, in appropriate hostcells.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. The amino acid sequence variants of the nucleic acids arepreferably constructed by mutating the polynucleotide to give an aminoacid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite.

Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

In a preferred method, polynucleotides encoding the novel nucleic acidsare changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).

PCR may also be used to create amino acid sequence variants of the novelnucleic acids. When small amounts of template DNA are used as startingmaterial, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thepolypeptide at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook, et al., supra, and CurrentProtocols in Molecular Biology, Ausubel, et al.

Due to the inherent degeneracy of the genetic code, other DNA 10sequences which encode substantially the same or a functionallyequivalent amino acid sequence may be used in the practice of theinvention for the cloning and expression of these novel nucleic acids.Such DNA sequences include those which are capable of hybridizing to theappropriate novel nucleic acid sequence under stringent conditions.

Furthermore, knowledge of the DNA sequence provided by the presentinvention allows for the modification of cells to permit, or increase,expression of endogenous CD39-like polypeptides. Cells can be modified(e.g., by homologous recombination) to provide increased CD39-likeexpression by replacing, in whole or in part, the naturally occurringCD39-like promoter with all or part of a heterologous promoter so thatthe cells express CD39-like polypeptides at a higher level. Theheterologous promoter is inserted in such a manner that it isoperatively linked to CD39-like encoding sequences. See, for example,PCT International Publication No. WO94/12650, PCT InternationalPublication No. WO92120808, and PCT International Publication No.WO91/09955. It is also contemplated that, in addition to heterologouspromoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and themultifunctional CAD gene which encodes carbamyl phosphate synthase,aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may beinserted along with the heterologous promoter DNA. If linked to theCD39-like coding sequence, amplification of the marker DNA by standardselection methods results in co-amplification of the CD39-like codingsequences in the cells.

The polynucleotides of the present invention also make possible thedevelopment, through, e.g., homologous recombination or knock outstrategies, of animals that fail to express functional CD39-likepolypeptides or that express a variant of a CD39-like polypeptide. Suchanimals are useful as models for studying the in vivo activities ofCD39-like polypeptides as well as for studying modulators of CD39-likepolypeptides.

6.4 Identification of Polymorphisms

Polymorphisms can be identified in a variety of ways known in the artwhich all generally involve obtaining a sample from a patient, analyzingDNA from the sample, optionally involving isolation or amplification ofthe DNA, and identifying the presence of the polymorphism in the DNA.For example, PCR may be used to amplify an appropriate fragment ofgenomic DNA which may then be sequenced. Alternatively, the DNA may besubjected to allele-specific oligonucleotide hybridization (in whichappropriate oligonucleotides are hybridized to the DNA under conditionspermitting detection of a single base mismatch) or to a singlenucleotide extension assay (in which an oligonucleotide that hybridizesimmediately adjacent to the position of the polymorphism is extendedwith one or more labelled nucleotides). In addition, traditionalrestriction fragment length polymorphism analysis (using restrictionenzymes that provide differential digestion of the genomic DNA dependingon the presence or absence of the polymorphism) may be performed.

Alternatively, a polymorphism resulting in a change in the amino acidsequence could also be detected by detecting a corresponding change inamino acid sequence of the protein, e.g., by an antibody specific to thevariant sequence.

6.5 Hosts

The present invention further provides host cells containing SEQ ID NO:1, 2, 24 or 26 of the present invention, wherein the nucleic acid hasbeen introduced into the host cell using known transformation,transfection or infection methods. The host cell can be a highereukaryotic host cell, such as a mammalian cell, a lower eukaryotic hostcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the recombinant construct intothe host cell can be effected by calcium phosphate transfection, DEAE,dextran mediated transfection, or electroporation (Davis, L., et al.,Basic Methods in Molecular Biology (1986)).

The host cells containing one of SEQ ID NO: 1, 2, 24 or 26 of thepresent invention, can be used in conventional manners to produce thegene product encoded by the isolated fragment (in the case of an ORF) orcan be used to produce a heterologous protein under the control of theEMF. Any host/vector system can be used to express one or more of theORFs of the present invention. These include, but are not limited to,eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular polypeptide or protein or which expresses the polypeptide orprotein at low natural level.

Mature proteins can be expressed in mammalian cells, yeast, bacteria,insect cells or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNA 10sequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements.

Recombinant polypeptides and proteins produced in bacterial culture areusually isolated by initial extraction from cell pellets, followed byone or more salting-out, aqueous ion exchange or size exclusionchromatography steps. Protein refolding steps can be used, as necessary,in completing configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

6.6 Peptides

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention.Fragments may be fused to carrier molecules such as immunoglobulins formany purposes, including increasing the valency of protein bindingsites. For example, fragments of the protein may be fused through“linker” sequences to the Fc portion of an immunoglobulin. For abivalent form of the protein, such a fusion could be to the Fc portionof an IgG molecule. Other immunoglobulin isotypes may also be used togenerate such fusions. For example, a protein-IgM fusion would generatea decavalent form of the protein of the invention. Analogs of thepolypeptides of the invention can be fused to another moiety ormoieties, e,g., targeting moiety or another therapeutic agent. Suchanalogs may exhibit improved properties such as activity and/orstability. By “degenerate variant” is intended nucleotide fragmentswhich differ from a nucleic acid fragment of the present invention(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of thegenetic code, encode an identical polypeptide sequence. Preferrednucleic acid fragments of the present invention are the ORFs whichencode proteins.

The invention also provides both full length and mature forms (forexample, without a signal sequence or precursor sequence) of CD39-likepolypeptides. The full length form of such proteins is identified in thesequence listing by translation of the nucleotide sequence of eachdisclosed clone. The mature form of such protein may be obtained byexpression of the full-length polynucleotide in a suitable mammaliancell or other host cell. The sequence of the mature form of the proteinis also determinable from the amino acid sequence of the full lengthform.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. This isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel, et al., CurrentProtocols in Molecular Biology.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The purified polypeptides are used in in vitro binding assays which arewell known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, forexample, small molecules, molecules from combinatorial libraries,antibodies or other proteins. The molecules identified in the bindingassay are then tested for antagonist or agonist activity in in vivotissue culture or animal models that are well known in the art. Inbrief, the molecules are titrated into a plurality of cell cultures oranimals and then tested for either cell/animal death or prolongedsurvival of the animal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to the tumor or othercell by the specificity of the binding molecule for SEQ ID NOs:3-4.

6.7 Gene Therapy

Mutations in the CD39-like gene that result in loss of normal functionof the CD39-like gene product underlie CD39-related human diseasestates. The invention comprehends gene therapy to restore CD39-likeactivity that would thus be indicated in treating those disease states.Delivery of a functional CD39-like gene to appropriate cells is effectedex vivo, in situ, or in vivo by use of vectors, and more particuarlyviral vectors (e.g., adenovirus, adeno-associated virus, or aretrovirus), or ex vivo by use of physical DNA transfer methods (e.g.,liposomes or chemical treatments). See, for example, Anderson, Nature,supplement to vol. 392, no 6679, pp. 25-30 (1998). For additionalreviews of gene therapy technology, see Friedmann, Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller,Nature, 357: 455460 (1992). Alternatively, it is contemplated that inother human disease states, preventing the expression of or inhibitingthe activity of CD39-like polypeptides will be useful in treating thedisease states. It is contemplated that antisense therapy or genetherapy could be applied to negatively regulate the expression ofCD39-like polypeptides.

6.8 Deposit of Clone

A plasmid containing DNA encoding the ACR III mutant was deposited withthe American Type Culture Collection (ATCC), 10801 University Avenue,Manassas, Va., on Jul. 13, 1999 under the terms of the Budapest Treaty(ATCC accession no. PTA-346).

6.9 Antibodies

In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., MonoclonalAntibodies Technology: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1984); St. Groth, et al., J. Immunol. 35:1-21 (1990);Kohler and Milstein, Nature 256:495497 (1975)), the trioma technique,the human B-cell hybridoma technique (Kozbor, et al., Immunology Today4:72 (1983); Cole, et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985), pp.77-96). In addition, techniques forpreparing chimeric and humanized antibodies (including polypeptidescontaining CDR and/or antigen-binding sequences of antibodies) are wellknown in the art.

Any animal (mouse, rabbit, etc.) which is known to produce antibodiescan be immunized with a peptide or polypeptide of the invention. Methodsfor immunization are well known in the art. Such methods includesubcutaneous or intraperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of the protein encodedby the ORF of the present invention used for immunization will varybased on the animal which is immunized, the antigenicity of the peptideand the site of injection.

The protein which is used as an immunogen may be modified oradministered in an adjuvant in order to increase the protein'santigenicity. Methods of increasing the antigenicity of a protein arewell known in the art and include, but are not limited to, coupling theantigen with a heterologous protein (such as globulin or -galactosidase)or through the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells.

Any one of a number of methods well known in the art can be used toidentify the hybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz, et al., Exp.Cell Research. 175:109-124 (1988)).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toproteins of the present invention.

For polyclonal antibodies, antibody containing antiserum is isolatedfrom the immunized animal and is screened for the presence of antibodieswith the desired specificity using one of the above-describedprocedures.

The present invention further provides the above-described antibodies indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, etc. Procedures for accomplishing such labeling arewell-known in the art, for example, see (Sternberger, L. A. et al., J.Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym.62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W.J. Immunol. Meth. 13:215 (1976)).

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whicha fragment of the polypeptide of interest is expressed. The antibodiesmay also be used directly in therapies or other diagnostics.

The present invention further provides the above-described antibodiesimmobilized on a solid support. Examples of such solid supports includeplastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, acrylic resins and polyacrylamide and latex beads.Techniques for coupling antibodies to such solid supports are well knownin the art (Weir, D. M. et al., “Handbook of Experimental Immunology”4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y.(1974)). The immobilized antibodies of the present invention can be usedfor in vitro, in vivo, and in situ assays as well as for immuno-affinitypurification of the proteins of the present invention.

6.10 Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magneticloptical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotidesequence information of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of dataprocessor structuring formats (e.g.text file or database) in order to obtain computer readable mediumhaving recorded thereon the nucleotide sequence information of thepresent invention.

By providing the nucleotide sequence of SEQ ID NO: 1, 2, 24 or 26, arepresentative fragment thereof, or a nucleotide sequence at least 99.9%identical to SEQ ID NO: 1, 2, 24 or 26 in computer readable form, askilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Software which implements the BLAST (Altschul,et al., J. Mol. Biol. 215:403410 (1990)) and BLAZE (Brutlag, et al.,Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system maybe used to identify open reading frames (ORFs) within a nucleic acidsequence. Such ORFs may be protein encoding fragments and may be usefulin producing commercially important proteins such as enzymes used infermentation reactions and in the production of commercially usefulmetabolites.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems is suitable for use inthe present invention.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein a nucleotidesequence of the present invention and the necessary hardware means andsoftware means for supporting and implementing a search means. As usedherein, “data storage means” refers to memory which can store nucleotidesequence information of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the nucleotidesequence information of the present invention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limited to,MacPattem (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisancan readily recognize that any one of the available algorithms orimplementing software packages for conducting homology searches can beadapted for use in the present computer-based systems.

As used herein, a “target sequence” can be any nucleic acid or aminoacid sequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein processing, may be of shorterlength.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

6.11 Expression Modulating Sequences

EMF sequences can be identified within a genome by their proximity tothe ORFs. An intergenic segment, or a fragment of the intergenicsegment, from about 10 to 200 nucleotides in length, taken 5′ from anyORF will modulate the expression of an operably linked 3′ ORF in afashion similar to that found with the naturally linked ORF sequence. Asused herein, an “intergenic segment” refers to the fragments of a genomewhich are between two ORF(S) herein described. Alternatively, EMFs canbe identified using known EMFs as a target sequence or target motif inthe computer-based systems of the present invention.

The presence and activity of an EMF can be confirmed using an EMF trapvector. An EMF trap vector contains a cloning site 5′ to a markersequence. A marker sequence encodes an identifiable phenotype, such asantibiotic resistance or a complementing nutrition auxotrophic factor,which can be identified or assayed when the EMF trap vector is placedwithin an appropriate host under appropriate conditions. As describedabove, an EMF will modulate the expression of an operably linked markersequence. A more detailed discussion of various marker sequences isprovided below. A sequence which is suspected of being an EMF is clonedin all three reading frames in one or more restriction sites upstreamfrom the marker sequence in the EMF trap vector. The vector is thentransformed into an appropriate host using known procedures and thephenotype of the transformed host is examined under appropriateconditions. As described above, an EMF will modulate the expression ofan operably linked marker sequence.

6.12 Triplex Helix Formation

In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee, et al., Nucl. Acids Res. 6:3073(1979); Cooney, et al., Science 15241:456 (1988); and Dervan, et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, FL (1988)).

Triple helix—formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques havebeen demonstrated to be effective in model systems. Informationcontained in the sequences of the present invention is necessary for thedesign of an antisense or triple helix oligonucleotide.

6.13 Diagnostic Assays and Kits

The present invention further provides methods to identify theexpression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies or one or more of nucleic acid probes of thepresent invention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells, or biological fluids such as sputum, blood,serum, plasma, or urine. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing protein extracts or membrane extracts ofcells are well known in the art and can be readily be adapted in orderto obtain a sample which is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartment kit to receive, inclose confinement, one or more containers which comprises: (a) a firstcontainer comprising one of the probes or antibodies of the presentinvention; and (b) one or more other containers comprising one or moreof the following: wash reagents, reagents capable of detecting presenceof a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe.

Types of detection reagents include labeled nucleic acid probes, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the enzymatic, or antibody binding reagents which are capableof reacting with the labeled antibody. One skilled in the art willreadily recognize that the disclosed probes and antibodies of thepresent invention can be readily incorporated into one of theestablished kit formats which are well known in the art.

6.14 Screening Assays

Using the isolated proteins of the present invention, the presentinvention further provides methods of obtaining and identifying agentswhich bind to a protein encoded by one of the ORFs from a nucleic acidwith a sequence of one of SEQ ID NO: 1, 2, 24 or 26, or to a nucleicacid with a sequence of one of SEQ ID NO: 1, 2, 24 or 26.

In detail, said method comprises the steps of: (a) contacting an agentwith an isolated protein encoded by one of the ORFs of the presentinvention, or nucleic acid of the invention; and (b) determining whetherthe agent binds to said protein or said nucleic acid.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention.

Alternatively, agents may be rationally selected or designed. As usedherein, an agent is said to be “rationally selected or designed” whenthe agent is chosen based on the configuration of the particularprotein. For example, one skilled in the art can readily adapt currentlyavailable procedures to generate peptides, pharmaceutical agents and thelike capable of binding to a specific peptide sequence in order togenerate rationally designed antipeptide peptides, for example seeHurby, et al., Application of Synthetic Peptides: Antisense Peptides,”In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp.289-307, and Kaspczak, et al., Biochemistry 28:9230-8 (1989), orpharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control.

One class of DNA binding agents are agents which contain base residueswhich hybridize or form a triple helix formation by binding to DNA orRNA. Such agents can be based on the classic phosphodiester, ribonucleicacid backbone, or can be a variety of sulfhydryl or polymericderivatives which have base attachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee, et al., Nucl. Acids Res. 6:3073(1979); Cooney, et al., Science 241:456 (1988); and Dervan, et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix—formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents.

Agents which bind to a protein encoded by one of the ORFs of the presentinvention can be used as a diagnostic agent, in the control of bacterialinfection by modulating the activity of the protein encoded by the ORF.Agents which bind to a protein encoded by one of the ORFs of the presentinvention can be formulated using known techniques to generate apharmaceutical composition.

6.15 Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from thenucleotide sequence of the SEQ ID NO: 1, 2, 24 or 26. Because thecorresponding gene is expressed in only one out of 18 tissues tested,namely macrophages, a hybridization probe derived from SEQ ID NO: 1, 2,24 or 26 can be used as an indicator of the presence of macrophage RNAin a sample. Any suitable hybridization technique can be employed, suchas, for example, in situ hybridization.

PCR as described U.S. Pat. Nos 4,683,195 and 4,965,188 providesadditional uses for oligonucleotides based upon the nucleotidesequences. Such probes used in PCR may be of recombinant origin, may bechemically synthesized, or a mixture of both. The probe will comprise adiscrete nucleotide sequence for the detection of identical sequences ora degenerate pool of possible sequences for identification of closelyrelated genomic sequences.

Other means for producing specific hybridization probes for nucleicacids include the cloning of nucleic acid sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.

The nucleotide sequences may be used to construct hybridization probesfor mapping their respective genomic sequences. The nucleotide sequenceprovided herein may be mapped to a chromosome or specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma, et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York N.Y. Fluorescent in situhybridization of chromosomal preparations and other physical chromosomemapping techniques may be correlated with additional genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScience (265:1981f). Correlation between the location of a nucleic acidon a physical chromosomal map and a specific disease (or predispositionto a specific disease) may help delimit the region of DNA associatedwith that genetic disease. The nucleotide sequences of the subjectinvention may be used to detect differences in gene sequences betweennormal, carrier or affected individuals.

The nucleotide sequence may be used to produce purified polypeptidesusing well known methods of recombinant DNA technology. Among the manypublications that teach methods for the expression of genes after theyhave been isolated is Goeddel, (1990) Gene Expression Technology,Methods and Enzymology, Vol 185, Academic Press, San Diego. Polypeptidesmay be expressed in a variety of host cells, either prokaryotic oreukaryotic. Host cells may be from the same species from which aparticular polypeptide nucleotide sequence was isolated or from adifferent species. Advantages of producing polypeptides by recombinantDNA technology include obtaining adequate amounts of the protein forpurification and the availability of simplified purification procedures.

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems and incorporated intothe INHERIT™670 Sequence Analysis System. In this algorithm, PatternSpecification Language (developed by TRW Inc., Los Angeles, Calif.) wasused to determine regions of homology. The three parameters thatdetermine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT™ 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul, S. F.,(1993) J Mol Evol 36:290-300; Altschul, S. F., et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

In addition, BLAST analysis was used to search for related moleculeswithin the libraries of the LIFESEQ™ database. This process, an“electronic northern” analysis is analogous to northern blot analysis inthat it uses one cellubrevin sequence at a time to search for identicalor homologous molecules at a set stringency. The stringency of theelectronic northern is based on “product score”. The product score isdefined as (% nucleotide or amino acid [between the query and referencesequences] in Blast multiplied by the % maximum possible BLAST score[based on the lengths of query and reference sequences]) divided by 100.At a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous or related molecules canbe identified by selecting those which show product scores betweenapproximately 15 and 30.

6.16 SEQ ID NOs:1-8, 23-24 and 26-27

Referring to FIG. 1, SEQ ID NO:1 is the nucleotide sequence of anexpressed sequence tag corresponding to a polynucleotide isolated from acDNA library of human fetal liver-spleen. SEQ ID NO:2 is an extendedversion of SEQ ID NO:1 obtained as described in Example 2, and theencoded polypeptide in SEQ ID NO: 3 is referred to herein as CD39-L4.SEQ ID NO:2 encodes a polypeptide having the amino acid sequence of SEQID NO:3 (shown in FIG. 2). The open reading frame corresponding to SEQID NO:3 starts at nucleotide 246, as numbered from the 5′ end of SEQ IDNO:2. This open reading frame encodes a polypeptide 428 amino acids inlength. The estimated molecular weight of the unglycosylated polypeptideis approximately 47.52 kDa.

Protein database searches with the BLAST algorithm indicate that SEQ IDNO:3 is homologous to the CD39 family. FIGS. 3A and 3B show the aminoacid sequence alignment between SEQ ID NO:3 (identified as “246 prot”)and human CD39 (“CD39Human.seq”), indicating that the two sequencesshare 30% amino acid sequence identity. Moreover, a higher degree ofhomology between the apyrase conserved regions (Kaczmarek et al., J.Biol. Chem. 271:33116-33122 (1996) is observed. In particular, an almostperfect match to a putative ATP-binding region was found from aminoacids 54-58, DAGST (DAGSS in CD39). In addition, the DLGGASTQ motif(DLGGASTQ in CD39), which is very well conserved among ATPDases, isfound from amino acids 199-206 in SEQ ID NO:3. Other regions conservedin apyrases were found from amino acids 129-134, ATAGLR (ATAGMR in CD39)and from amino acids 169-173, GSDEG (GQEEG in CD39).

SEQ ID NO:3 differs from CD39 in that SEQ ID NO:3 contains a hydrophobicstretch of 22 amino acids at its amino terminus, which is indicative ofa leader peptide. SEQ ID NO:3 also lacks the transmembrane domain foundat the carboxyl terminus of CD39. These features indicate that SEQ IDNO:3 is a soluble ATPDase.

SEQ ID NO:3 shares an even higher degree of homology (83% identity) witha murine NTPase, as shown in the amino acid sequence alignment presentedin FIGS. 4A and 4B (SEQ ID NO:3 is identified as “246 prot,” and mouseCD39 as “mur ntpase”).

The message encoding SEQ ID NO:3 is tightly regulated in atissue-specific manner. An expression study using a semiquantitativePCR/Southern blot approach revealed a significant level of expression inmacrophage. In contrast, human CD39 is expressed in tissues such asplacenta, lung, skeletal muscle, kidney, and heart.

SEQ ID NO: 4 is the polynucleotide sequence for CD39-L4. SEQ ID NO: 5 isthe corresponding amino acid sequence.

SEQ ID NO: 6 is the polynucteotide sequence for a CD39-L4 variantdesignated ACRIII, wherein the following amino acid substitutions havebeen made: D168→T, S170→Q and L175→F; SEQ ID NO: 7 is the correspondingamino acid sequence.

SEQ ID NO: 8 is the genomic sequence for the human CD39-L4 gene; exonsappear at nucleotides 1-288 (exon 1), 1281-1580 (exon 2), 1820-1855(exon 3) 2467-2555 (exon 4), 2863-2942 (exon 5), 3889-3950 (exon 6),48944995 (exon 7), 5847-5987 (exon 8), 6966-7138 (exon 9) and 8556-9365(exon 10).

SEQ ID NO: 24 is the polynucleotide sequence for a CD39-L4 splicevariant that creates an isoform designated CD39-L66. SEQ ID NO: 25 isthe corresponding amino acid sequence.

SEQ ID NO: 26 is the polynucleotide sequence for CD39-L2. SEQ ID NO: 27is the corresponding amino acid sequence.

6.17 Uses of Novel CD39-Like Polypeptides and Antibodies

Polypeptides of the invention having ATPDase, including NDPase, activityare useful for inhibiting platelet function and can therefore beemployed in the prophylaxis or treatment of pathological conditionscaused by or involving thrombosis or excessive coagulation or excessiveplatelet aggregation, such as myocardial infarction, cerebral ischemia,angina, and the like. Polypeptides of the invention can also be used inthe maintenance of vascular grafts. Platelet function can be measured byany of a number of standard assays, such as, for example, the plateletaggregation assay described in Example 5.

Such pathological conditions include conditions caused by or. involvingarterial thrombosis, such as coronary artery thrombosis and resultingmyocardial infarction, cerebral artery thrombosis or intracardiacthrombosis (due to, e.g., atrial fibrillation) and resulting stroke, andother peripheral arterial thrombosis and occlusion; conditionsassociated with venous thrombosis, such as deep venous thrombosis andpulmonary embolism; conditions associated with exposure of the patient'sblood to a foreign or injured tissue surface, including diseased heartvalves, mechanical heart valves, vascular grafts, and otherextracorporeal devices such as intravascular cannulas, vascular accessshunts in hemodialysis patients, hemodialysis machines andcardiopulmonary bypass machines; and conditions associated withcoagulapathies, such as hypercoagulability and disseminatedintravascular coagulopathy. Co-administration of other agents suitablefor treating the pathological condition, e.g., other anti-coagulationagents, is also contemplated.

In particular, variants like the ACRIII mutant described herein areexpected to be superior therapeutics for treating such pathologicalconditions because (1) ACRIII exhibits six-fold greater activitycompared to wild type CD39-L4, and (2) ACRIII, like CD39-L4, is uniquelyspecific for ADP and does not hydrolyze ATP. Thus, adverse side effectsfrom hydrolysis of circulating ATP are avoided.

For instance, ATP is known to act as an extracellular signal in manytissues. In the heart, extracellular ATP modulates ionic processes andcontractile function (for review see Burnstock, G., Neuropharmacology36:1127). Recently, it has been shown that extracellular ATP markedlyinhibits glucose transport in rat cardiomyocytes (Fisher, Y. et al., J.Biol. Chem. 274:755-761. Another source of extracellular ATP is thatreleased from parenchymal cells under hypoxic or ischemic conditions(Skobel, E., and Kammermeier, H. Biochim. Biophys. Acta 1362:128-134).ATP is also involved in the modulation of anti-IgE-induced release ofhistamine from human lung mast cells (Schulman, E. S., et al., Am. J.Respir. Cell Mol. Biol. 20:520-537).

Furthermore, the ability of CD39-L4 to hydrolyze NDPs other than ADP hasimplications outside the circulatory system. For instance, it has beenreported that UDP is the most potent agonist for the human P2Y₆receptor. Communi, et al., Bioch Bioph Res Com 222:303-308 (1996). Thisreceptor is expressed in several tissues including infiltrating T cellspresent in inflammatory bowel disease. Somers, et al., Lab Invest78:1375-1383 (1998). In this microenvironment, a molecule with theenzymatic properties of CD39-L4 could influence T cell responses bymodifying the extracellular half-life of UDP. Another role for CD39-L4has been suggested by the report that mouse CD39-L4 maps closely to alocus associated with audigenic brain seizures in mice. See Chadwick, etal., Genomics 50:357-367 (1998); Seyfried, et al., Genetics 99:117-126(1981). This locus, known as Asp-1, is thought to be linked or tocorrespond to a factor that influences Ca²⁺-ATPase activity. Neumann, etal., Behav. Genetics 20:307-323 (1990).

Additionally, the polypeptides of the invention can be used as molecularweight markers, and as a food supplement. A polypeptide consisting ofSEQ ID NO:3, for example, has a molecular mass of approximately 47.52 kDin its unglycosylated form. Protein food supplements are well known andthe formulation of suitable food supplements including polypeptides ofthe invention is within the level of skill in the food preparation art.

The polypeptides of the invention are also useful for making antibodysubstances that are specifically immunoreactive with CD39-like proteins.Antibodies and portions thereof (e.g., Fab fragments) which bind to thepolypeptides of the invention can be used to identify the presence ofsuch polypeptides in a sample. For example, the level of the nativeprotein corresponding to SEQ ID NO:3 in a blood sample can be determinedas an indication of vascular condition. Such determinations are carriedout using any suitable immunoassay format, and any polypeptide of theinvention that is specifically bound by the antibody can be employed asa positive control.

Additionally, the polypeptides of the invention are useful formodulating the ratios of levels of adenosine molecules in vivo toregulate homeostasis. Adenosine diphosphate (ADP) is an agonist ofplatelet activation and aggregation. It has been demonstrated that theP2Y receptor (and others including P2T and P2Y1 and potentially others)transduces this signal. Adenosine triphosphate (ATP) also binds to thisreceptor, but acts as an antagonist. Therefore, the ratios of levels ofATP/ADP can significantly influence in vivo platelet activation andaggregation. Agents that specifically decrease levels of ADP not onlydecrease the amount of agonist available to signal, but also increasethe relative antagonistic effects of ATP, because of less competitionfor the common receptor.

The polypeptides of the invention are administered by any route thatdelivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. For treatment of vascular disease, polypeptides according to theinvention are generally administered intravenously. In vivo murinestudies with soluble human CD39 have shown that mice injectedintravenously with 50 mg recombinant soluble human CD39 in 100 mlsterile saline had biologically active CD39 in their sera for anextended period of time, with an elimination half-life of almost 2 days(Gayle, R. B., et al., J. Clinical Invest. 101:1851-1859 (1998)).Suitable dosage ranges for the polypeptides of the invention can beextrapolated from these dosages or from similar studies in appropriateanimal models. Dosages can then be adjusted as necessary by theclinician to provide maximal therapeutic benefit.

6.18 Pharmaceutical Formulations and Routes of Administration

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be administered to a patient in need, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to treat or ameliorate a variety of disorders.Such a composition may also contain (in addition to protein and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF,Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.

The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or compliment its activity oruse in treatment. For example, CD39-L2 or CD39-L4 may be co-adnsteredwith platelet ADP receptor antagonists, e.g. ATP derivatives, ADPderivatives. Such additional factors and/or agents may be included inthe pharmaceutical composition to produce a synergistic effect withprotein of the invention, or to minimize side effects. Conversely,protein of the present invention may be included in formulations of theparticular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent tominimize side effects of the cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent. A protein of the present invention may be active in multimers(e.g., heterodimers or homodimers) or complexes with itself or otherproteins. As a result, pharmaceutical compositions of the invention maycomprise a protein of the invention in such multimeric or complexedform.

Techniques for formulation and administration of the compounds of theinstant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

6.18.1. Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein of the present invention used in the pharmaceuticalcomposition or to practice the method of the present invention can becarried out in a variety of conventional ways, such as oral ingestion,inhalation, topical application or cutaneous, subcutaneous,intraperitoneal, parenteral or intravenous injection. Intravenousadministration to the patient is preferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops.Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

6.18.2. Compositions/formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein of the present invention is administered orally, protein of thepresent invention will be in the form of a tablet, capsule, powder,solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carriersuch as a gelatin or an adjuvant. The tablet, capsule, and powdercontain from about 5 to 95% protein of the present invention, andpreferably from about 25 to 90% protein of the present invention. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin such as peanut oil, mineral oil, soybeanoil, or sesame oil, or synthetic oils may be added. The liquid form ofthe pharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of protein of the present invention, andpreferably from about 1 to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.Dragee cores are providedwith suitable coatings. For this purpose, concentrated sugar solutionsmay be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.Dyestuffs or pigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentratedsolutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.1 μg to about 1 mg) of protein of thepresent invention per kg body weight. For compositions of the presentinvention which are useful for bone, cartilage, tendon or ligamentregeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein of theinvention which may also optionally be included in the composition asdescribed above, may alternatively or additionally, be administeredsimultaneously or sequentially with the composition in the methods ofthe invention. Preferably for bone and/or cartilage formation, thecomposition would include a matrix capable of delivering theprotein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins ofthe invention may be combined with other agents beneficial to thetreatment of the bone and/or cartilage defect, wound, or tissue inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), transforminggrowth factors (TGF-.alpha. and TGF-.beta.), and insulin-like growthfactor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention. The dosage regimen of aprotein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins, e.g., amount oftissue weight desired to be formed, the site of damage, the condition ofthe damaged tissue, the size of a wound, type of damaged tissue (e.g.,bone), the patient's age, sex, and diet, the severity of any infection,time of administration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and with inclusion ofother proteins in the pharmaceutical composition. For example, theaddition of other known growth factors, such as IGF I (insulin likegrowth factor I), to the final composition, may also effect the dosage.Progress can be monitored by periodic assessment of tissue/bone growthand/or repair, for example, X-rays, histomorphometric determinations andtetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA). Cells may alsobe cultured ex vivo in the presence of proteins of the present inventionin order to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo for therapeuticpurposes.

6.18.3. Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of the C-proteinase activity). Such information can be usedto more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the C-proteinase inhibiting effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; for example, the concentration necessaryto achieve 50-90% inhibition of the C-proteinase using the assaysdescribed herein. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

6.18.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition.

The present invention is illustrated in the following examples. Uponconsideration of the present disclosure, one of skill in the art willappreciate that many other embodiments and variations may be made in thescope of the present invention. Accordingly, it is intended that thebroader aspects of the present invention not be limited to thedisclosure of the following examples.

EXAMPLE 1 Isolation of SEQ ID NO:1 from a cDNA Library of Human FetalLiver-Spleen

A plurality of novel nucleic acids were obtained from a b2HFLS20W cDNAlibrary prepared from human fetal liver-spleen, as described in Bonaldoet al., Genome Res. 6:791-806 (1996), using standard PCR, Sequencing byhybridization sequence signature analysis, and Sanger sequencingtechniques. The inserts of the library were amplified with PCR usingprimers specific for vector sequences flanking the inserts. Thesesamples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. One of these inserts was identifiedas a novel sequence not previously obtained from this library and notpreviously reported in public databases. This sequence is shown in FIG.1 as SEQ ID NO:1.

EXAMPLE 2 Isolation of SEQ ID NO:2 and Determination of a NucleotideSequence Encoding a 428-Amino Acid Protein with Sequence Homology toCD39

The nucleotide sequence shown in FIG. 1, and labeled SEQ ID NO:2,encodes the translated amino acid sequence SEQ ID NO:3, which is shownin FIG. 2. The extended nucleotide sequence was obtained by isolatingcolonies generated from pools of clones from a human macrophage cDNAlibrary (Invitrogen, Cat. #A550-25). Briefly, the macrophage cDNAlibrary was plated on LB/Amp plates (containing 100 mg/ml ampicillin) ata density of about 40,000 colonies/plate. The colonies were lifted ontonitrocellulose filters and hybridized with a radiolabeled probegenerated from the original clone (i.e., SEQ ID NO:1).

That the identified clones corresponded to SEQ ID NOs:1 and 2 wasconfirmed by using gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′[SEQ ID NO: 9] and 5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) in aPCR-based assay. Then PCR using vector- and gene-specific primers wasemployed to amplify the 5′ portion of the cDNA. Nested primers were usedto generate sequence from the amplified product(s). Laser gene™ softwarewas used to edit and “contig” the partial sequences into a full-lengthsequence. As discussed above, the amino acid sequence has strikinghomology to CD39, which is involved in modulating platelet reactivityduring vascular inflammation. Based in part on the observed sequencesimilarity to CD39, the polypeptide encoded by SEQ ID NO: 2 wasdesignated CD39-L4.

EXAMPLE 3

A. Expression of SEQ ID NOS. 3 and 5 in COS-7 Cells

COS-7 cells were grown in DMEM (ATCC) and 10% fetal bovine serum (FBS)(Gibco) to 70% confluence. Prior to transfection the media was changedto DMEM and 0.5% FCS. Cells were transfected with cDNAs for SEQ ID NOs.3 and 5 or with pBGaI vector by the FuGENE-6 transfection reagent(Boehringer). In summary, 4 μl of FuGENE-6 was diluted in 100 μl of DMEMand incubated for 5 minutes. Then, this was added to 1 μg of DNA andincubated for 15 minutes before adding it to a 35 mm dish of COS-7cells. The COS-7 cells were incubated at 37 ° C. with 5% CO₂. After 24hours, media and cell lysates were collected, centrifuged and dialyzedagainst assay buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mMCaCl₂ and MgCl₂. More robust expression can be achieved using theprotocol described in Example 6 below.

B. Expression Study Using SEQ ID NO:2

The expression of SEQ ID NO. 2 in various tissues was analyzed using asemi-quantitative polymerase chain reaction-based technique. Human cDNAlibraries were used as sources of expressed genes from tissues ofinterest (adult brain, adult heart, adult kidney, adult lymph node,adult liver, adult lung, adult ovary, adult placenta, adult spleen,adult testis, bone marrow, fetal kidney, fetal liver, fetalliver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).Gene-specific primers (5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-GCAGGTCTCCAAGGAAGTACG-3′ [SEQ ID NO: 11 ]) were used to amplifyportions of the SEQ ID NO:2 sequence from the samples. Amplifiedproducts were separated on an agarose gel, transferred and chemicallylinked to a nylon filter. The filter was then hybridized with aradioactively labeled (α³³P-dCTP) double-stranded probe generated fromthe full-length SEQ ID NO:2 sequence using a Klenow polymerase,random-prime method. The filters were washed (high stringency) and usedto expose a phosphorimaging screen for several hours. Bands indicatedthe presence of cDNA including SEQ ID NO:2 sequences in a specificlibrary, and thus mRNA expression in the corresponding cell type ortissue.

Of the 18 human tissues tested, macrophage was the only sample thatprovided a signal, indicating that expression of SEQ ID NO:2 is tightlyregulated. In contrast, the CD39 molecule has been found in tissues suchas placenta, lung, skeletal muscle, kidney and heart.

EXAMPLE 4 Chromosomal Localization of the Gene Corresponding to SEQ IDNOs:1 and 2

Chromosome mapping technologies allow investigators to link genes tospecific regions of chromosomes. Assignment to chromosome 14 wasperformed with the Coriell cell repository monochromosomal panel #2(NIGMS cell repository). This human rodent somatic cell hybrid panelconsists of DNA isolated from 24 hybrid cell cultures retaining 1 humanchromosome each. The panel was screened with gene-specific primers(5′-GCTACCTCACTTCCTTTGAG-3′ [SEQ ID NO: 9] and5′-CTGGCTGGTGAAGTTTTCCTC-3′ [SEQ ID NO: 10]) that generated a sequencetag site (STS). The Genebridge 4 radiation hybrid panel was alsoscreened (Research Genetics), and the results of the PCR screening weresubmitted to the Whitehead/MIT Radiation Hybrid mapping email server athttp://www-genome.wi.mit.edu.

EXAMPLE 5 Platelet Aggregation Assay

Blood is anticoagulated with 0.1 volume 3.2% sodium citrate.Platelet-rich plasma (PRP) is prepared with an initial whole bloodcentrifugation (200×g, 15 min., 25° C.) and a second centrifugation ofthe PRP (90×g, 10 min.) to eliminate residual erythrocytes andleukocytes. The stock suspension of PRP is maintained at roomtemperature under 5% CO₂-air. The platelet aggregation assay uses atwo-sample, four-channel Whole Blood Lumi-Aggregometor, model 560(Chronolog Corp., Havertown, Pa.). PRP containing 1.22×108 platelets ispreincubated with the sample to be tested for inhibition of aggregationfor 10 min. at 37° C. in a siliconized glass cuvette containing astirring bar, followed by stimulation with either ADP (5 mm), collagen(5 mg/ml), or thrombin (0.1 unit/ml). Platelet aggregation is recordedfor at least 10 min. Data are expressed as the percentage of lighttransmission with platelet-poor plasma equal to 100%.

EXAMPLE 6 CD39-L4 is a Soluble Apyrase

The mammalian ectoapyrase CD39 is an integral membrane protein with twotransmembrane domains (one at each end of the protein) (Maliszewski, C.R. et al., J. Immunol. 153:3574-3583). The hydrophobicity profiles forthe deduced amino acid sequence of other family members, such as CD39L1and CD39L3, are very similar to CD39 (Chadwick, B. P. and Frischauf A.M., Genomics 50:357-367), suggesting that these proteins also have twomembrane spanning domains. However, CD39-L4 does not appear to have asecond transmembrane domain at its C-terminus, suggesting that theN-terminus hydrophobic region could code for a secretory signal. To testthis hypothesis, CD39-L4 was subcloned into the mammalian expressionvector pCDNA3.1 and a 6-Histidine tag was inserted into the codingsequence.

The CD39-L4 cDNA sequence was initially isolated from a macrophage cDNAlibrary (Invitrogen). The sense primer(5′-TTAAAGCTTGGGAAAAGAATGGCCACTTC-3′, SEQ ID NO. 20) with a HindIII siteand the antisense primer (5′-AGACTCGAGGTGGCTCAATGGGAGATGCC-3′, SEQ IDNO. 21) with a XhoI site were used to subclone the coding sequences intothe mammalian expression vector pcDNA3.1 (Invitrogen). The nucleotidesequence of the insert is set forth in SEQ ID NO. 4. In order toimmunologically detect the protein, the coding region was furthermodified so that it would include a Gly-Ser-6His epitope tag immediatelyfollowing Arg²⁴. Briefly, two partially overlapping complementaryoligonucleotides (5′-GCGCTGTCTCCCACAGAGGATCGCATCACCATCACCATCACAACCAGCAGACTTGGTT-3′ (SEQ ID. NO. 22) and5′-AACCAAGTCTGCTGGTTGTGATGGTGATGGTGATGCGATCCTCTGTGGG AGACAGCGC-3′ (SEQID NO. 23)) were used on the CD39-L4 pcDNA3.1 template. The primers wereextended in opposite directions around the plasmid using a 12 cycle PCRprogram (95° C., 1 minute; 60° C., 1 minute; 72° C., 15 minutes)(Stratagene). The reaction was treated with DpnI to digest themethylated parental DNA and then transformed into E. coli. Colonies werescreened for the insert.

To ascertain whether CD39-L4-6His is secreted, the coding region of theCD39-L4-6His protein was inserted into the pcDNA3.1 expression vectorand transiently transfected into COS-7 cells. Cos-7 cells obtained fromthe American Tissue Type Culture Collection were grown in DMEMsupplemented with 10% FBS and 100 units/ml penicillin G and 100streptomycin sulfate at 37° C. in 10% CO₂. Transfections were performedat 75% confluency in 10 cm plates with Fugene-6 according to themanufacturers instructions. The cells in 7 mls of medium were incubatedwith 16 μl of Fugene-6 and 8 μg of DNA for 14-18 hours. At the end ofthe transfection the medium was replaced with DMEM medium containing lowserum (1% FBS). The cells were then incubated for 24-48 hours prior toharvesting.

The CD39-L4-6His was concentrated by treating the cell lysates andmedium with Nickel-NTA agarose (Qiagen) followed by SDS/PAGE andimmunoblot analysis with an antibody against the Arg-Gly-Ser-6Hisepitope. Cells were washed twice with PBS containing 0.5 μg/mlleupeptin, 0.7 μg/ml pepstatin and 0.2 μg/ml aprotinin. After a briefsonication and centrifugation step to clear the lysate, the samples werethen incubated with a Nickel-NTA resin at 4° C. for 2-3 hours. Thehistidine-tagged protein complexed to the resin was washed three timeswith PBS before loading onto a 10% SDS/PAGE gel for Western blotanalysis. CD39-L4 was detected in both the cell lysate and the mediumfrom cells transfected with the CD39-L4-6His expression vector, but notfrom control cells. While the predicted molecular weight of CD39-L4-6Hisis 46 kDa, the immunoreactive protein exhibited a mobility by SDS/PAGEcorresponding to a molecular mass of approximately 51 kDa in the mediaand approximately 48 kDa in the cell lysate. The difference in apparentmolecular weight may be due to posttranslational modications of threepotential N-glycosylation sites in the CD39-L4 predicted amino acidsequence.

Secretion of CD39-L4 was also examined by treatment of the transfectedcells with brefeldin A, an inhibitor of translocation of secretoryproteins from the endoplasmic reticulum to the Golgi apparatus.Chadwick, et al., Genomics 50:357-367 (1998). Brefeldin A was dissolvedin ethanol and added to the transfected cells 48 hours aftertransfection. Both control and brefeldin A treated cells were washedonce with PBS and incubated for 8 hours in medium with none or varyingdosages of brefeldin A. Increasing dosages of brefeldin A blockedsecretion of CD39-L4-6His and led to massive intracellular accumulation.

EXAMPLE 7 Assay for ATPase Activity

Apyrase activity was determined by measuring the amount of [³³P]P_(i)released from [γ³³P]ATP. In summary, 50 μl of samples were incubated inthe presence of 10 μCi of [γ³³P]ATP for one hour at 37 ° C. The[³³P]P_(i) released and the [γ³³P]ATP were separated by thin layerchromatography (TLC) plates (EM Science). The solvent system consistedof 1 M KH₂PO₄. The separated compounds were scanned for radioactivitywith a Phosphoimager (Molecular Dynamics, Sunnyvale, Calif.) andquantitated by ImageQuant software. COS-7 cells transfected with SEQ IDNOs. 3 and 25 had at least a four fold increase in activity over cellstransfected with the vector alone. Although ATPase activity was present,Example 13 demonstrates that CD39-L4 has significantly more NDPaseactivity.

EXAMPLE 8 Site-directed Mutagenesis of CD39L4

Site directed mutagenesis was employed to increase the enzymaticactivity of CD39L4. Amino acid sequence comparisons between CD39 familymembers reveal four highly homologous regions in all five human members(Chadwick and Frischauf, Genomics 50:357-367, 1998). These regions,termed apyrase-conserved regions (ACRs), are present not only in theCD39 family members but other apyrases from species as distant as yeastand plants. Examination of similarities and differences in the CD39 ACRsled to the design of three CD39L4 mutants (see FIG. 5). In thesemutants, codons encoding CD39 ACR specific residues were used to replacecodons from the CD39L4 wild type ACR sequence. Only residues withsignificantly different structural or chemical properties were replaced.A PCR based approach was used to produce these mutations.

Briefly, the expression vector pCDNA3.1 (Invitrogen) containing the fullcoding sequence of the CD39L4 gene (with a 6 Histidine tag insertedafter Arg 24 in the coding sequence to allow purification of thesecreted mature form of the protein) was subjected to a PCR-basedsite-directed mutagenesis approach using overlapping oligonucleotides[CD39-L4 ACR I mutant (nt 177-148 and 160-204): 5′-GTG AGT GCT CCC TGCATC TAA CAT MT TCC-3′ (SEQ ID NO: 12) and 5′-GAT GCA GGG AGC ACT CAC ACTAGT ATT CAT GTT TAC ACC TTT GTG-3′ (SEQ ID NO: 13); CD39-L4 ACR IImutant (nt 402-359 and 385415): 5′-GCG TAG TCC TGC TGT TGC CCC TAG GTACAC TGG GGT CTT TTT CC-3′ (SEQ ID NO: 14) and 5′-GCA ACA GCA GGA CTA CGCTTA CTG CCA GAA C-3′ (SEQ ID NO: 15); and CD39-L4 ACR III mutant (nt532485 and 513-540): 5′-CCC MG CGA ATA TGC CTT CGT CTT GTC CAG TCA TGATGC TAA CAC TGC-3′ (SEQ ID NO: 16) and 5′-CGA AGG CAT ATT CGC TTG GGTTAC TGT G-3′ (SEQ ID NO:17)]. After amplification of the whole plasmidwith Pfu DNA polymerase (Stratagene) (95° C./1 min; 60° C./1 min; 72°C./15 min for 12 cycles), the methylated parental DNA was digested withthe restriction enzyme DpnI, leaving only the unmethylated PCR amplifiedproducts. The resulting annealed double-stranded nicked products werethen transformed into bacteria and the resulting colonies were screenedfor the desired mutations by sequencing. The subsequent constructs werefully sequenced to verify that the mutations were in fact introduced andthat no extraneous mutations were generated.

EXAMPLE 9 ACR III Mutant Increases ADPase Activity

Plasmids containing the mutated and wild type forms of the CD39L4 genewere transfected into COS-7 cells. After two days, protein was purifiedfrom the culture medium using a Nickel-NTA resin approach to concentratethe tagged proteins. These proteins were then assayed for ATPase andADPase activity by measuring the inorganic phosphate released (Wang, T.F., et al., J. Biol. Chem. 273:24814-24821, 1998). The proteins wereincubated in apyrase buffer (15 mM Tris pH 7.4, 135 mM NaCl, 2mM EGTAand 10 mM glucose) for 1 hour at 37 ° C. with or without 2 mM CaCl₂ or 2mM MgCl₂. Phosphatase reactions were initiated by the addition of ADP orATP to a final concentration of 1 mM. The reaction of inorganicphosphorus with ammonium molybdate in the presence of sulfuric acid,produces an unreduced phosphomolybdate complex. The absorbance of thiscomplex at 340 nm is directly proportional to the inorganic phosphorusconcentration (Daly, J. A., and Ertingshausen G., Clin. Chem. 18:263(1972) (Sigma Diagnostics)).

As seen in FIG. 7, mutations in ACR I and II eliminate activity, whereasthe mutations in ACR III increase activity six-fold over wild type. Thisincreased activity therefore offers a greater therapeutic potential, asless protein could be administered to offer the same pharmacologicaleffect. The replacement of three amino acids in the III region (aminoacids 167 to 181 in CD39-L4) and the resulting increase in ADPaseactivity predicts that replacement of additional amino acids within thisregion by amino acids from the equivalent region of CD39 may alsoenhance the activity of the protein over wild type CD39L4. The increasein ADPase activity over wild type may also be due to the replacement ofonly one or two of the three amino acids; this can be confirmed byreplacing one or two amino acids at a time.

The polynucleotide and amino acid sequences of a CD39-L4 variant termedACRIII and having the amino acid substitutions D168→T, S170→Q and L175→Fcompared to wild type CD39-L4 (SEQ ID NO: 5) are set forth in SEQ IDNOs: 6 and 7, respectively, and in FIG. 6.

EXAMPLE 10 ACR III Mutant and Wild Type Forms are Specific for ADP andnot ATP

Both the CD39L4 wild type and the CD39L4 variant with mutations in theACRIII region hydrolyze ADP. Hover, when ATP was tested as a substrate,neither the CD39L4 nor the CD39L4 mutant, ACR III, catalyzed hydrolysis.In contrast, CD39 as a membrane bound molecule (Marcus, et al., TheJournal of Clinical Investigation, 99: 1351-1360) or as a geneticallyengineered soluble form (Gayle, et al., The Journal of ClinicalInvestigation, 101:1851-1858,1998) is able to hydrolyze both ATP and ADPsubstrates efficiently. The specificity that both CD39L4 wild type andthe CD39L4 ACR III mutant have for ADP is an advantageous feature thatmakes these CD39L4-type molecules better antiplatelet therapeuticcandidates than CD39, as ADP is the agonist that causes plateletaggregation. Therapeutics that have both ADPase and ATPase activitiespotentially could create adverse side effects by interfering with levelsof ATP in the circulation.

EXAMPLE 11 Organization of the Human CD39-L4 Gene

A human CITB BAC genomic library (Research Genetics) was screened withgene specific primers [246-I6 (nt 5522-5543),5′-CTTCCTTCACTGGGAATTCAGG-3′ (SEQ ID NO: 18) and 246-K4 (nt 49224945),5′-CTGTTTACCGAGATGGTTGGAAGC-3′ (SEQ ID NO: 19)] using a PCR based assay.

Briefly, gene specific primers were used to screen pools of BAC DNAs.BAC pools that produced an amplified DNA fragment of the predicted sizewere pursued until an individual BAC was identified. BAC63-118 wasisolated and sequenced with gene specific primers for the CD39-L4 cDNA,as well as intron specific primers. The CD39-L4 coding sequence wasfound to be distributed over 10 exons spanning 9.3 kb of genomic DNA asset out in SEQ ID NO: 8.

EXAMPLE 12 CD39-L4 and CD39-L2 are Stimulated by Divalent Cations

The high degree of conservation in the apyrase conserved regions ofCD39-L4 and CD39-L2 suggests similar function to other apyrases. To testthis hypothesis, COS-7 cells were transfected with the CD39-L4-6His andCD39-L2myc-His construct as described herein. The medium fromtransfected cells was incubated with Nickel-NTA resin (Qiagen) in orderto capture the 6His tagged protein, the resin was washed with assaybuffer (buffer A, 15 mM Tris pH 7.5, 134 mM NaCl and 5 mM glucose) andthe protein still tethered to the resin in a suspension was assayed forADPase activity. Nucleotidase activity was determined by measuring theamount of inorganic phosphate released from nucleotide substrates usingthe technique of Dlay and Ertingshausen, Clin. Chem. 18:263-265 (1972).In this reaction the complex of inorganic phosphorus with phosphorreagent (ammonium molybdate in the presence of sulfuric acid) producesan unreduced phosphomolybdate compound. The absorbance of this complexat 340 nm is directly proportional to the inorganic phosphorusconcentration. The protein still tethered to the resin as a 30% (50% forCD39-L2) suspension in buffer A was assayed by the addition of thenucleotide to a final concentration of 1 mM and incubated at 37° C. for30 minutes. The reaction was stopped by adding 100 volumes of phosphorreagent. The amount of phosphate released from the reaction wasquantified using a calcium/phosphorus combined standard (Sigma). Theamount of protein used in the assays was estimated by comparing theintensity of the bands in Western blots with that of a series ofstandards of known quantity. CD39-L4 protein from transfected cellsdisplayed a 2.3 fold increase in activity over the cells transfectedwith the vector alone. When Ca²⁺ and Mg²⁺ were added, the activityincreased 3.6 fold and 6 fold, respectively. CD39-L2 protein fromtransfected cells displayed an 8.7 fold increase in activity over thecells transfected with the vector alone. When Ca²⁺ and Mg²⁺ were added,the activity of the CD39-L2 cells increased another 2-3 fold.

EXAMPLE 13 Characterization of CD39-L4 Activity

CD39-L4 protein was assayed for ADPase activity in the presence ofdifferent kinds of inhibitors of ADPases. Control ecto-apyrase activitywas determined with protein tethered to the Nickel-NTA resin. Bothassays were performed as described above except the protein was inbuffer A containing 2 mM CaCl₂ and 2 mM MgCl₂. As shown by Table 1below, inhibitors of phosphatases (F⁻) and adenylate kinase (Ap5A) didnot inhibit activity. The inhibitors of vacuolar ATPases (NEM),mitochondrial ATPases (N3⁻) and Na⁺, K⁺, ATPase (ouabain) did notsignificantly inhibit the Ca²⁺ and Mg²⁺ stimulated activity. However,metal chelators (EDTA and EGTA) significantly inhibited activity. Theseresults show that the overwhelming majority of the activity in theassays originates from a protein bound to the resin with characteristicsof an E-type apyrase.

TABLE 1 Inhibition of CD39-L4 activity INHIBITORS % OF CONTROL Control100 ± 7  Ouabain (1 mM) 96 ± 6  NEM (10 mM) 106 ± 5  N3⁻ (1 mM) 100 ±12  F⁻ (10 mM) 113 ± 5  Ap5A (10 μM) 121 ± 9  EGTA (2 mM) 35 ± 3  EDTA(2 mM) 52 ± 3 

As shown in Table 2 below, the nucleotide specificity of CD39-L4 wasalso assayed as described above. The CD39-L4 activity was determinedwith protein tethered to the Ni-NTA resin. The protein was in buffer Acontaining 1 mM EGTA, as well as 2 mM CaCl₂ and MgCl₂. The assay wasstarted by adding the nucleotides to a final concentration of 1 mM. Thevalues below-are expressed relative to ADP. The relative activity of thenucleotide triphosphates varies almost seven-fold with ATP being thepoorest substrate. No phosphate release was detected with AMP and ADPwas hydrolyzed at a rate approximately twenty-fold higher than ATP. Theother nucleotide diphosphates (GDP and UDP) were also very efficientlyhydrolyzed by CD39-L4. These results indicate that CD39-L4 defines a newclass of E-type apyrase in humans with a specificity for NDPs asenzymatic substrates.

TABLE 2 Substrate specificity of CD39-L4 NUCLEOTIDE % OF CONTROL ADP 100± 15  ATP 5 ± 1 AMP 0 CTP 26 ± 2  GTP 34 ± 1  UTP 12 ± 4  CDP 268 ± 11 GDP 334 ± 38  UDP 408 ± 14 

EXAMPLE 14 Glycosylation is not Essential for CD39-L4 Activity

Posttranslational modifications such as N-linked glycosylation arecommon in secreted and membrane-bound mammalian proteins. Thesemodifications may be important for correct protein folding or enzymaticactivity and are not easily reproduced when the proteins are expressedin other organisms such as bacteria. In order to test whether CD39-L4 isglycosylated, COS-7 cells, transfected as described in Example 6, weretreated with tunicamycin (Sigma), which blocks the formation ofN-glycosidic linkages.

COS-7 cells were grown to 75% confluency and transfected with theCD39-L4-6His construct. After 24 hours, a fraction of the COS-7 cellswere treated with Tunicamycin at a concentration of 5 μg/ml. The mediawas replaced again after 24 hours with fresh tunicamycin and harvestedafter 48 hours. The CD39-L4-6His protein was concentrated by treatingthe media with Nickel-NTA agarose (Qiagen). The resin was washed withassay buffer and the protein still tethered to the resin in a suspensionwas assayed for a shift in electrophoretic mobility as well as itsADPase activity.

Western blot analysis using an antibody against the 6-His epitoperevealed that the glycosylated CD39-L4 protein isolated from the controlcells had an approximate size of 51 kDa. However, tunicamycin treatedcells had a molecular weight of approximately 46 kDa indicating that theprotein was deglycosylated.

ADPase activity of the tunicamycin treated cells was assayed asdescribed in Example 12 above. The deglycosylated CD39-L4 protein hadADPase activity comparable to an equal amount of the glycosylatedprotein isolated from control cells. This demonstrates thatglycosylation of the protein is not important for ADPase activity.

EXAMPLE 15 Cloning and Expression of CD39-L2

The CD39-L2 coding sequence (SEQ ID NO: 26) was subcloned intopcDNA3.1/myc-His(+)A (Invitrogen) via the EcoRI and XbaI sites. Briefly,a human adult heart cDNA library (Gibco BRL) was subjected to polymerasechain reaction (PCR) using gene-specific primers L2-5′B(5′-CGTATCCCGCGGGTGGAGGCCGGGGTG-3′, SEQ ID NO: 28) and L2-3′B(5′-CTTCTGCAAGTCCCAGAGCCAGTGTGC-3′, SEQ ID NO: 29). The resultingproducts were diluted 100-fold and subjected to a second round of PCRwith primers L2-5′A (5′-GGAGCCCAAAAGACCGGCTGC-3′, SEQ ID NO: 30) andL2-3′A (5′-TGAAGTCACGTCCAGGACAGG-3′, SEQ ID NO: 31). The productrepresented a single band by agarose gel and was purified and sequencedto confirm its identity. Primers corresponding to the translationalstart region and the carboxy terminal region, excluding the stop codon,of the CD39-L2 coding sequence, L2EcoMet(5′-CGGAATTCAACATGAAAAAAGGTAATCCGTTATGAA-3′, SEQ ID NO: 32) and L2Xba3′(5′-TGTCTAGATGAGGCTGGACTCTTCTG-3′, SEQ ID NO: 33) were used on thepurified DNA to produce a DNA fragment corresponding to the entirecoding region of the CD39-L2 gene, flanked by EcoRI and Xbal sites. ThisPCR product was digested to generate overhang ends that were ligatedinto the EcoRI and XbaI sites of pcDNA3.1/myc-His(+)A. The resultingplasmid allowed expression of the CD39-L2 coding sequence fused in framewith the myc-6His epitope at the carboxy terminus.

Transfection of COS-7 cells was performed as described below. COS-7cells obtained from the American Tissue Type Culture Collection weregrown in MDEM supplemented with 10% FBS and 100 units/ml penicillin Gand 100 μg/ml streptomycin sulfact at 37° C. in 10% CO₂. Transfectionswere performed at 75% confluency in 10 cm plates with Fugene-6 accordingto the manufacturer's instructions. The cells in 10 ml of medium wereincubated with 16 μl of Fugene-6 and 8 μg of DNA for 48 hours. Themedium was then replaced by DMEM containing low serum (1% FBS), andincubated for 48 hours before harvesting.

EXAMPLE 16 Cellular Localization of CD39-L2

Western blot analysis was performed on COS cells transfected with theplasmid described in Example 15 above to determine the cellularlocalization of CD39-L2. To detect myc epitope tagged recombinantproteins, the blot was incubated with a 2000-fold dilution of theanti-c-myc monoclonal antibody (Invitrogen) at room termperature for twohours. The secondary antibody (anti-mouse Ig AP conjugate) was diluted1000-fold and incubated for 1-2 hours at room temperature. Boundantibody was detected by using Sigma Fast™5-bromo4-chloro-3indolylphosphate/nitro blue tetrazolium (BCIP/NTP) as the alkaline phosphatasesubstrate according to instructions of the manufacturer.

The recombinant protein was detected in the media and the membranefractions of the CD39-L2 transfected cells, but not in the cytosolicfraction or control transfections. The relative band intensities suggestthat the majority of the recombinant CD39-L2 protein is secreted intothe media and a fraction resides in the membrane. The predictedmolecular weight of unprocessed CD39-L2 is 53 kD. However, the membraneand secreted fractions displayed slower mobility by SDS/PAGE than thatpredicted by its amino acid content, suggesting post translationalmodification.

To confirm that recombinant CD39-L2 is secreted, the cellularlocalization was performed using increasing amounts of brefeldin A, aninhibitor of translocation of secretory proteins from the endoplasmicreticulum to the Golgi apparatus. Recombinant CD39-L2 in the mediadecreased in a brefeldin A dose dependent manner. Recombinant CD39-L2also accumulated in the cytosol in a dose dependent manner. Therefore,recombinant CD39-L2 secretion follows the conventional cellularsecretory pathway.

Flow cytometric analysis was used to determine if recombinant CD39-L2 isexpressed on cell surfaces. COS-7 cells were transfected as describedabove with either pcDNA3.1/myc-His(+)A or pCD39-L2myc-HIS. After 72hours of transfection, the cells were washed twice with PBS, anddislodged with 10 mM EDTA in PBS. Cells were pelleted by centrifugationat 300 g for five minutes, washed with PBS and resuspended in bindingbuffer (PBS containing 3% FBS and 0.02% sodium azide) at a concentrationof 1×10⁶ cells per 100 μl. The cells were first stained with 20 μg/ml ofmonoclonal anti-myc antibody for 30 minutes at 4° C. The cells were thenwashed with binding buffer and stained with 20 μg/ml of R-phycoerythrinconjugated goat anti-mouse IgG antibody (Molecular Probes, Eugene,Oreg.). After washing with binding buffer, the cells were resuspended in1 ml of binding buffer and analyzed on the FACScalibur flow cytometer(Becton Dickinson Immunocytometry Systems, San Jose, Calif.).

Expression of cell surface recombinant CD39-L2 was found only on cellstransfected with pCD39-L2myc-His, while cells from the controltransfection showed no antibody binding. These results confirm thatCD39-L2 is a secreted apyrase.

EXAMPLE 17 Characterization of CD39-L2 Activity

CD39-L2 protein was assayed for ADPase activity in the presence ofdifferent kinds of inhibitors of ADPases. Control ecto-apyrase activitywas determined with protein tethered to the Nickel-NTA resin. Bothassays were performed as described in Example 12 above except theprotein was in buffer A containing 1 mM EGTA and 3mM CaCl₂. The assaywas started by adding ADP to 1 mM followed by a 30 minute incubation at37° C. As shown by Table 3 below, CD39-L2 is not inhibited by inhibitorsof vacuolar adenosine triphospatase (ATPases) (NEM), mitochondrialATPase (N₃ ⁻) and Na⁺, K⁺ ATPase (oubain). An inhibitor of adenylatekinase (Ap5A) did not inhibit activity, while an inhibitor ofphosphatases (F⁻) partially inhibited activity. Metal chelators (EDTAand EGTA) inhibited CD39-L2 activity thereby demonstrating that CD39-L2activity is dependent on divalent cations.

TABLE 3 Inhibition of CD39-L2 activity INHIBITORS % OF CONTROL Control100 ± 3  Ouabain (1 mM) 101 ± 9  NEM (10 mM) 88.4 ± 13   N₃ ⁻ (1 mM) 90± 13 F⁻ (10 mM) 63 ± 9  Ap5A (10 mM) 87 ± 11 EGTA (2 mM) 34 ± 10 EDTA (2mM) 18.4 ± 9  

As shown in Table 4 below, the nucleotide specificity of CD39-L2 wasalso assayed as described in Example 12. The CD39-L2 activity wasdetermined with protein tethered to the Ni-NTA resin. The protein was inassay buffer A containing 1 mM EGTA, 3 mM CaCl₂ and 3 mM MgCl₂. Theassay was started by adding the nucleotides to a final concentration of1 mM. The values are expressed relative to ADP. The samples were assayedat 37° C. for 30 minutes.

TABLE Substrate Specificity of CD39-L2 NUCLEOTIDE % OF CONTROL ADP 100 ±8  ATP 16 ± 2  AMP 0.6 ± 1   CTP 44 ± 4  GTP 39 ± 1  UTP 13 ± 1  CDP 282± 18  GDP 338 ± 52  UDP 303 ± 5 

These results confirm that CD39-L2 along with CD39-L4 define a new classof E-type apyrase in humans with a specificity for NDPs as enzymaticsubstrates.

EXAMPLE 18 CD39-L4 and CD39-L2 Expression Using In Situ Hybridization

A. In Situ Hybridization of CD39-L4 in Kidney

A 298 nt fragment of the CD39L4 cDNA 3′-untranslated region wasamplified by PCR with oligonucleotide primers 246D13 and 246D4(5′-ATCCTGGACTTGAGCCTAGAG-3′, SEQ ID NO: 34 and5′-CTGATATTGATGGGTCTTGGG-3′, SEQ ID NO: 35). The fragment was subclonedinto the pCR™ II-TOPO plasmid (Invitrogen) and sense and antisense RNAwere synthesized. The probe was labeled using the digoxigenin labelingkit supplied by Boehringer-Mannheim as described in the manufacturersprotocol. Automated in situ hybridization was performed by Qua MolecularLabs (Santa Barbara, Calif.) using a modified version of a previouslypublished procedure (Myers, J. A., et al., (1995) J Surg. Path. 1,191-203). The Ventana Medical Systems, Inc. (Tucson, Ariz.) TechMate™Automated Staining System was used for this procedure. All tissues werefixed in 10% neutral buffered formalin, paraffin-embedded and cut into 4μm thick sections. Sections were placed onto Ventana's ChemMate™Capillary Gap Slides (POP075).

Staining of kidney sections revealed that specific cell types hybridizedwith the antisense probe but not the sense probe in a highly specificmanner. The staining of the glomerulus revealed that the epithelia ofthe Bowman's capsule, podocyte epithelia and mesangial cells werespecifically stained. The expression of the CD39L4 protein in thisregion could be necessary to prevent platelet aggregation in theBowman's capsule because platelets become highly concentrated in thisparticular region as water and ions are filtered from the blood. Thebloody region within the kidney showed staining of white blood cells,presumably macrophages. This staining is consistent with previousstudies where a macrophage cDNA library showed expression of the CD39L4cDNA. CD39L4 staining was also found in some tubule epithelial cells inthe kidney.

B. In Situ Hybridization of CD39-L2 in Heart

A 186 nt fragment of the CD39L2 cDNA was amplified by PCR witholigonucleotide primers L2RNA3 and L2RNA2 (5′-GGATGGAAAGGAGTTGGTCAG-3′,SEQ ID NO: 36 and 5′-GTCCACATGCTTCACTTCCTC-3′ SEQ ID NO: 37). Thefragment was subcloned into the pCR™ II-TOPO plasmid (Invitrogen) andsense and antisense RNA were synthesized and labeled as described above.Automated in situ hybridization was performed as described above.

Staining of heart sections revealed that specific cell types hybridizedwith the antisense probe but not the sense probe in a highly specificmanner. The cardiac muscle cells as well as capillary endothelial cellsand white blood cells within a blood vessel showed specific staining.This staining is consistent with previous studies where a heart cDNAlibrary showed robust expression of the CD39L2 cDNA.

This in situ hybridization data is consistent with a physiological rolefor CD39-L4 and CD39-L2 in regulating platelet aggregation andhemostasis. Further in situ hybridization may be carried out to confirmthis activity.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and compositions and methods which are functionallyequivalent are within the scope of the invention. Indeed, numerousmodifications and variations in the practice of the invention areexpected to occur to those skilled in the art upon consideration of thepresent preferred embodiments. Consequently, the only limitations whichshould be placed upon the scope of the invention are those which appearin the appended claims. All references cited within the body of theinstant specification are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210> SEQ ID NO 1 <211>LENGTH: 300 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:1 ggcatattag cttgggttac tgtgaatttt ctgacaggtc agctgcatgg ccacagacag 60gagactgtgg ggaccttgga cctaggggga gcctccaccc aaatcacgtt cctgccccag 120tttgagaaaa ctctggaaca aactcctagg ggctacctca cttcctttga gatgtttaac 180agcacttata agctctatac acatagttac ctgggatttg gattgaaagc tgcaagacta 240gcaaccctgg gagccctgga gacagaaggg actgatgggc acactttccg gagtgcctgt 300<210> SEQ ID NO 2 <211> LENGTH: 1799 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(246)..(1529) <221> NAME/KEY: misc_feature <222> LOCATION: (1718) <223>OTHER INFORMATION: n = adenine or guanine or cytosine or thymine <400>SEQUENCE: 2 gcgggctgcc gcgcaagggt ggcgcgcgcg cgttttcctt gttcctggtcaacaaagaaa 60 tgtggagtgt cttggctgaa tcctcataca gacaagatca ttatggtgctgttaggttga 120 aaaagtgata taataaagga accaaggaga aaattcagaa ggaaagaaaaaattgcctct 180 gcaggtgtgc gagcaggatt gcttctgcaa caaaagcctc cacccagccacatcttggga 240 aaaga atg gcc act tct tgg ggc aca gtc ttt ttc atg ctg gtggta tcc 290 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser1 5 10 15 tgt gtt tgc agc gct gtc tcc cac agg aac cag cag act tgg tttgag 338 Cys Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu20 25 30 ggt atc ttc ctg tct tcc atg tgc ccc atc aat gtc agc gcc agc acc386 Gly Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr 3540 45 ttg tat gga att atg ttt gat gca ggg agc act gga act cga att cat434 Leu Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His 5055 60 gtt tac acc ttt gtg cag aaa atg cca gga cag ctt cca att cta gaa482 Val Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu 6570 75 ggg gaa gtt ttt gat tct gtg aag cca gga ctt tct gct ttt gta gat530 Gly Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp 8085 90 95 caa cct aag cag ggt gct gag acc gtt caa ggg ctc tta gag gtg gcc578 Gln Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala 100105 110 aaa gac tca atc ccc cga agt cac tgg aaa aag acc cca gtg gtc cta626 Lys Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu 115120 125 aag gca aca gca gga cta cgc tta ctg cca gaa cac aaa gcc aag gct674 Lys Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys Ala 130135 140 ctg ctc ttt gag gta aag gag atc ttc agg aag tca cct ttc ctg gta722 Leu Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val 145150 155 cca aag ggc agt gtt agc atc atg gat gga tcc gac gaa ggc ata tta770 Pro Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu 160165 170 175 gct tgg gtt act gtg aat ttt ctg aca ggt cag ctg cat ggc cacaga 818 Ala Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg180 185 190 cag gag act gtg ggg acc ttg gac cta ggg gga gcc tcc acc caaatc 866 Gln Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile195 200 205 acg ttc ctg ccc cag ttt gag aaa act ctg gaa caa act cct aggggc 914 Thr Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly210 215 220 tac ctc act tcc ttt gag atg ttt aac agc act tat aag ctc tataca 962 Tyr Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr225 230 235 cat agt tac ctg gga ttt gga ttg aaa gct gca aga cta gca accctg 1010 His Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu240 245 250 255 gga gcc ctg gag aca gaa ggg act gat ggg cac act ttc cggagt gcc 1058 Gly Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg SerAla 260 265 270 tgt tta ccg aga tgg ttg gaa gca gag tgg atc ttt ggg ggtgtg aaa 1106 Cys Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly ValLys 275 280 285 tac cag tat ggt ggc aac caa gaa ggg gag gtg ggc ttt gagccc tgc 1154 Tyr Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu ProCys 290 295 300 tat gcc gaa gtg ctg agg gtg gta cga gga aaa ctt cac cagcca gag 1202 Tyr Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu His Gln ProGlu 305 310 315 gag gtc cag aga ggt tcc ttc tat gct ttc tct tac tat tatgac cga 1250 Glu Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr AspArg 320 325 330 335 gct gtt gac aca gac atg att gat tat gaa aag ggg ggtatt tta aaa 1298 Ala Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly IleLeu Lys 340 345 350 gtt gaa gat ttt gaa aga aaa gcc agg gaa gtg tgt gataac ttg gaa 1346 Val Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp AsnLeu Glu 355 360 365 aac ttc acc tca ggc agt cct ttc ctg tgc atg gat ctcagc tac atc 1394 Asn Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu SerTyr Ile 370 375 380 aca gcc ctg tta aag gat ggc ttt ggc ttt gca gac agcaca gtc tta 1442 Thr Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser ThrVal Leu 385 390 395 cag ctc aca aag aaa gtg aac aac ata gag acg ggc tgggcc ttg ggg 1490 Gln Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp AlaLeu Gly 400 405 410 415 gcc acc ttt cac ctg ttg cag tct ctg ggc atc tcccat tgaggccacg 1539 Ala Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His420 425 tacttccttg gagacctgca tttgccaaca cctttttaag gggaggagagagcacttagt 1599 ttctgaacta gtctggggac atcctggact tgagcctaga gattwrgttaattaascggc 1659 cgagcttatc cttwatragg taatttactt gcmtggccgc gtttacacgtcgtgatggna 1719 aacctgcgtc ccaactaacg cttgasamat ccccttcgca gctgcgataccaaaagccga 1779 cgacgccttc cacagtgcca 1799 <210> SEQ ID NO 3 <211>LENGTH: 428 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:3 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 1015 Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 2530 Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 4045 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 5560 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 7075 80 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 8590 95 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys100 105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val LeuLys 115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala LysAla Leu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro PheLeu Val Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser AspGlu Gly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly GlnLeu His Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu GlyGly Ala Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys ThrLeu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met PheAsn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly PheGly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu ThrGlu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro ArgTrp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln TyrGly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 AlaGlu Val Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315320 Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325330 335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val340 345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu GluAsn 355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser TyrIle Thr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser ThrVal Leu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr GlyTrp Ala Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly IleSer His 420 425 <210> SEQ ID NO 4 <211> LENGTH: 1287 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(1284) <400> SEQUENCE: 4 atg gcc act tct tgg ggc aca gtcttt ttc atg ctg gtg gta tcc tgt 48 Met Ala Thr Ser Trp Gly Thr Val PhePhe Met Leu Val Val Ser Cys 1 5 10 15 gtt tgc agc gct gtc tcc cac aggaac cag cag act tgg ttt gag ggt 96 Val Cys Ser Ala Val Ser His Arg AsnGln Gln Thr Trp Phe Glu Gly 20 25 30 atc ttc ctg tct tcc atg tgc ccc atcaat gtc agc gcc agc acc ttg 144 Ile Phe Leu Ser Ser Met Cys Pro Ile AsnVal Ser Ala Ser Thr Leu 35 40 45 tat gga att atg ttt gat gca ggg agc actgga act cga att cat gtt 192 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr GlyThr Arg Ile His Val 50 55 60 tac acc ttt gtg cag aaa atg cca gga cag cttcca att cta gaa ggg 240 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu ProIle Leu Glu Gly 65 70 75 80 gaa gtt ttt gat tct gtg aag cca gga ctt tctgct ttt gta gat caa 288 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser AlaPhe Val Asp Gln 85 90 95 cct aag cag ggt gct gag acc gtt caa ggg ctc ttagag gtg gcc aaa 336 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu GluVal Ala Lys 100 105 110 gac tca atc ccc cga agt cac tgg aaa aag acc ccagtg gtc cta aag 384 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro ValVal Leu Lys 115 120 125 gca aca gca gga cta cgc tta ctg cca gaa cac aaagcc aag gct ctg 432 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys AlaLys Ala Leu 130 135 140 ctc ttt gag gta aag gag atc ttc agg aag tca cctttc ctg gta cca 480 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro PheLeu Val Pro 145 150 155 160 aag ggc agt gtt agc atc atg gat gga tcc gacgaa ggc ata tta gct 528 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 tgg gtt act gtg aat ttt ctg aca ggt cag ctgcat ggc cac aga cag 576 Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu HisGly His Arg Gln 180 185 190 gag act gtg ggg acc ttg gac cta ggg gga gcctcc acc caa atc acg 624 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala SerThr Gln Ile Thr 195 200 205 ttc ctg ccc cag ttt gag aaa act ctg gaa caaact cct agg ggc tac 672 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln ThrPro Arg Gly Tyr 210 215 220 ctc act tcc ttt gag atg ttt aac agc act tataag ctc tat aca cat 720 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr LysLeu Tyr Thr His 225 230 235 240 agt tac ctg gga ttt gga ttg aaa gct gcaaga cta gca acc ctg gga 768 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala ArgLeu Ala Thr Leu Gly 245 250 255 gcc ctg gag aca gaa ggg act gat ggg cacact ttc cgg agt gcc tgt 816 Ala Leu Glu Thr Glu Gly Thr Asp Gly His ThrPhe Arg Ser Ala Cys 260 265 270 tta ccg aga tgg ttg gaa gca gag tgg atcttt ggg ggt gtg aaa tac 864 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile PheGly Gly Val Lys Tyr 275 280 285 cag tat ggt ggc aac caa gaa ggg gag gtgggc ttt gag ccc tgc tat 912 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val GlyPhe Glu Pro Cys Tyr 290 295 300 gcc gaa gtg ctg agg gtg gta cga gga aaactt cac cag cca gag gag 960 Ala Glu Val Leu Arg Val Val Arg Gly Lys LeuHis Gln Pro Glu Glu 305 310 315 320 gtc cag aga ggt tcc ttc tat gct ttctct tac tat tat gac cga gct 1008 Val Gln Arg Gly Ser Phe Tyr Ala Phe SerTyr Tyr Tyr Asp Arg Ala 325 330 335 gtt gac aca gac atg att gat tat gaaaag ggg ggt att tta aaa gtt 1056 Val Asp Thr Asp Met Ile Asp Tyr Glu LysGly Gly Ile Leu Lys Val 340 345 350 gaa gat ttt gaa aga aaa gcc agg gaagtg tgt gat aac ttg gaa aac 1104 Glu Asp Phe Glu Arg Lys Ala Arg Glu ValCys Asp Asn Leu Glu Asn 355 360 365 ttc acc tca ggc agt cct ttc ctg tgcatg gat ctc agc tac atc aca 1152 Phe Thr Ser Gly Ser Pro Phe Leu Cys MetAsp Leu Ser Tyr Ile Thr 370 375 380 gcc ctg tta aag gat ggc ttt ggc tttgca gac agc aca gtc tta cag 1200 Ala Leu Leu Lys Asp Gly Phe Gly Phe AlaAsp Ser Thr Val Leu Gln 385 390 395 400 ctc aca aag aaa gtg aac aac atagag acg ggc tgg gcc ttg ggg gcc 1248 Leu Thr Lys Lys Val Asn Asn Ile GluThr Gly Trp Ala Leu Gly Ala 405 410 415 acc ttt cac ctg ttg cag tct ctgggc atc tcc cat tga 1287 Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His420 425 <210> SEQ ID NO 5 <211> LENGTH: 428 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Ala Thr Ser Trp Gly Thr ValPhe Phe Met Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser HisArg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met CysPro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp AlaGly Ser Thr Gly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys MetPro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser ValLys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala GluThr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro ArgSer His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala GlyLeu Arg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe GluVal Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 LysGly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu Ala 165 170 175Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185190 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195200 205 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr210 215 220 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr ThrHis 225 230 235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu AlaThr Leu Gly 245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr PheArg Ser Ala Cys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile PheGly Gly Val Lys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu ValGly Phe Glu Pro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg GlyLys Leu His Gln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe TyrAla Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met IleAsp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu ArgLys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser GlySer Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu LeuLys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 LeuThr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 420 425 <210> SEQ ID NO6 <211> LENGTH: 1287 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1284) <400> SEQUENCE:6 atg gcc act tct tgg ggc aca gtc ttt ttc atg ctg gtg gta tcc tgt 48 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15gtt tgc agc gct gtc tcc cac agg aac cag cag act tgg ttt gag ggt 96 ValCys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 atcttc ctg tct tcc atg tgc ccc atc aat gtc agc gcc agc acc ttg 144 Ile PheLeu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 tat ggaatt atg ttt gat gca ggg agc act gga act cga att cat gtt 192 Tyr Gly IleMet Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60 tac acc tttgtg cag aaa atg cca gga cag ctt cca att cta gaa ggg 240 Tyr Thr Phe ValGln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 gaa gtt tttgat tct gtg aag cca gga ctt tct gct ttt gta gat caa 288 Glu Val Phe AspSer Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 cct aag cag ggtgct gag acc gtt caa ggg ctc tta gag gtg gcc aaa 336 Pro Lys Gln Gly AlaGlu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 gac tca atc ccccga agt cac tgg aaa aag acc cca gtg gtc cta aag 384 Asp Ser Ile Pro ArgSer His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 gca aca gca ggacta cgc tta ctg cca gaa cac aaa gcc aag gct ctg 432 Ala Thr Ala Gly LeuArg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 ctc ttt gag gtaaag gag atc ttc agg aag tca cct ttc ctg gta cca 480 Leu Phe Glu Val LysGlu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 aag ggc agtgtt agc atc atg act gga caa gac gaa ggc ata ttc gct 528 Lys Gly Ser ValSer Ile Met Thr Gly Gln Asp Glu Gly Ile Phe Ala 165 170 175 tgg gtt actgtg aat ttt ctg aca ggt cag ctg cat ggc cac aga cag 576 Trp Val Thr ValAsn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 gag act gtgggg acc ttg gac cta ggg gga gcc tcc acc caa atc acg 624 Glu Thr Val GlyThr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205 ttc ctg ccccag ttt gag aaa act ctg gaa caa act cct agg ggc tac 672 Phe Leu Pro GlnPhe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 ctc act tccttt gag atg ttt aac agc act tat aag ctc tat aca cat 720 Leu Thr Ser PheGlu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 agt tacctg gga ttt gga ttg aaa gct gca aga cta gca acc ctg gga 768 Ser Tyr LeuGly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 gcc ctggag aca gaa ggg act gat ggg cac act ttc cgg agt gcc tgt 816 Ala Leu GluThr Glu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 tta ccgaga tgg ttg gaa gca gag tgg atc ttt ggg ggt gtg aaa tac 864 Leu Pro ArgTrp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 cag tatggt ggc aac caa gaa ggg gag gtg ggc ttt gag ccc tgc tat 912 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 gcc gaagtg ctg agg gtg gta cga gga aaa ctt cac cag cca gag gag 960 Ala Glu ValLeu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320 gtccag aga ggt tcc ttc tat gct ttc tct tac tat tat gac cga gct 1008 Val GlnArg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 gttgac aca gac atg att gat tat gaa aag ggg ggt att tta aaa gtt 1056 Val AspThr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 gaagat ttt gaa aga aaa gcc agg gaa gtg tgt gat aac ttg gaa aac 1104 Glu AspPhe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 ttcacc tca ggc agt cct ttc ctg tgc atg gat ctc agc tac atc aca 1152 Phe ThrSer Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 gccctg tta aag gat ggc ttt ggc ttt gca gac agc aca gtc tta cag 1200 Ala LeuLeu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400ctc aca aag aaa gtg aac aac ata gag acg ggc tgg gcc ttg ggg gcc 1248 LeuThr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415acc ttt cac ctg ttg cag tct ctg ggc atc tcc cat tga 1287 Thr Phe His LeuLeu Gln Ser Leu Gly Ile Ser His 420 425 <210> SEQ ID NO 7 <211> LENGTH:428 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Thr Gly Gln Asp GluGly Ile Phe Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly TrpAla Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly Ile SerHis 420 425 <210> SEQ ID NO 8 <211> LENGTH: 9365 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: exon <222>LOCATION: (1)..(288) <221> NAME/KEY: exon <222> LOCATION: (1281)..(1580)<221> NAME/KEY: exon <222> LOCATION: (1820)..(1855) <221> NAME/KEY: exon<222> LOCATION: (2467)..(2555) <221> NAME/KEY: exon <222> LOCATION:(2863)..(2942) <221> NAME/KEY: exon <222> LOCATION: (3889)..(3950) <221>NAME/KEY: exon <222> LOCATION: (4894)..(4995) <221> NAME/KEY: exon <222>LOCATION: (5847)..(5987) <221> NAME/KEY: exon <222> LOCATION:(6966)..(7138) <221> NAME/KEY: exon <222> LOCATION: (8556)..(9365) <221>NAME/KEY: misc_feature <222> LOCATION: (3409) <223> OTHER INFORMATION: n= a or g or t or c <221> NAME/KEY: misc_feature <222> LOCATION: (9214)<223> OTHER INFORMATION: n = a or g or t or c <221> NAME/KEY:misc_feature <222> LOCATION: (9303) <223> OTHER INFORMATION: n = a or gor t or c <221> NAME/KEY: misc_feature <222> LOCATION: (9311) <223>OTHER INFORMATION: n = a or g or t or c <400> SEQUENCE: 8 gcctctgcaggtgtgcgagc aggattgctt ctgcaacaaa agcctccacc cagccacatc 60 ttgggaaaagaatggccact tcttggggca cagtcttttt catgctggtg gtatcctgtg 120 tttgcagcgctgtctcccac aggaaccagc agacttggtt tgagggtatc ttcctgtctt 180 ccatgtgccccatcaatgtc agcgccagca ccttgtatgg aattatgttt gatgcaggga 240 gcactggaactcgaattcat gtttacacct ttgtgcagaa aatgccaggt aagtgcaact 300 gggrcccttagtagagtctg taaatccaca ctttagcatc tcctcccaga aacaaatatg 360 ctgagagtttattatgtgaa ttacagaatc tcacacctag tggatgtctt tcttcagaga 420 actttggactacaattgaac atgtgggtta tttatttatt tttatttatt tgttttgttt 480 ttattttttaactttttttt tgagacaagg tcttgctttg ttgcccggtc tgtagtgcag 540 tggcatgatgacacatcact gcaaccttga cctcctgggc tcaagcagtc cttccacctc 600 agccccctgagttgttgaga ctacaggctt gtgccaccat gcccagctca tttttaaatt 660 tttttatagagacctgctca gactggcctc aaactcctag gctcaattga tcctcccacc 720 tcagcctcccaaagtactgg gattataggt gtaagtcacc atgcttggcc agaacacatg 780 gcttaattcaatgtgaaatt agaagagagc tgggctgtct gtagtctgaa acccatgtgt 840 tcaaaaagaatagttataat ttgttcttcc tctttaaaca tgggatactc cagggatcca 900 taatattcagaatatgggga gtggttttgg gagaaggatc acatgagaat ttcactgcca 960 tccttggacatgaggctagg aatccctgaa gattaacttt ttctgaattt gtcagtgttt 1020 tttcctcaggtcacttatgg agcctgggga aaggtggagg agttaggtgt ccaccagaga 1080 aatggtagcagaaatggacc ctcagaggtt gctctagtcc ttctttccag tactcctgca 1140 agacattcctcacaactagg atcattgggg taacttcagg gaagtcatag gaaaacttac 1200 agagacagagcccagcatct gaagcagcct aacttttggt aaccagctct ctcttctgtt 1260 ttgttccatgracaaaatag gacagcttcc aattctagaa ggggaagttt ttgattctgt 1320 gaagccaggactttctgctt ttgtagatca acctaagcag ggtgctgaga ccgttcaagg 1380 gctcttagaggtggccaaag actcaatccc ccgaagtcac tggaaaaaga ccccagtggt 1440 cctaaaggcaacagcaggac tacgcttact gccagaacac aaagccaagg ctctgctctt 1500 tgaggtaaaggagatcttca ggaagtcacc tttcctggta ccaaagggca gtgttagcat 1560 catggatggatccgacgaag gtgggagagg tgttgatatg cgttccaggg ggagaggggc 1620 aggatcagtgaaagatctaa ctaaaggaac tggggccagg aataaacaga aggaatgaga 1680 tagcaggaaatagaagacag ggagaaggga acatgtgctc tagacatgga atttagagag 1740 gaaaaaaaaaaaacaaggtt ggggccagga aagagaaaaa atgctctggg atctaatcct 1800 tgtctttctttctttttagg catattagct tgggttactg tgaattttct gacaggtaat 1860 acatcctcaagtttatcttt agagcttaac tagcttttac atgcatagtc agaggagtaa 1920 aagcctcttctttcattctg tattgtttct tcttctttaa aaaaggaaaa gaggctgggt 1980 gtggcagttcatgcctgtta attccagcgc tttgggaggc tgagttgggc agatcacttg 2040 aggccaggagttcaagacca gcctggccaa catggcgaaa ctccgtctct accaaaaata 2100 caaaaatagctgggcatggt ggtgtgtacc tgtagtccca gctactcagg aggctggaga 2160 atcacttgaacccaggaggc agaggttgca gtgagctgag agccgagatt gcgccactgc 2220 actccaggctggatgataga gcaagactct gtctccaaaa aggccttcca aaaaaaaaaa 2280 aaacacctgccttgaaggcc tctgctgcaa caagagtcct tccgagttga cattcacctg 2340 cagccttggggctggggagc agtggagtat atatggaata ccttcagtgt atgataagag 2400 caagagagacaagtgttggg ctgcccagga tgtcgaggct atttagagct ggctctcatt 2460 tgacaggtcagctgcatggc cacagacagg agactgtggg gaccttggac ctagggggag 2520 cctccacccaaatcacgttc ctgccccagt ttgaggtgag tcatttaatg aagatctggt 2580 tagaagtgcacttggcaggc gtatcatggt gccaagaaag aggcgcccca ttttcagcca 2640 gcagctctaccacgcttagg cagagtcaag tcaattaata actaggtgaa tgttcccttg 2700 ccatctcactgttcagaatc ccttcgtttc ctcaagccta gtgagattag ccccttaatc 2760 tgtcttcatctctgattttt tgctgggagg gacgggtggt ggtgtgaaca tcttcaggta 2820 attacagatcctgaatagct ttttgctttt tctgatttgc agaaaactct ggaacaaamt 2880 cyatrgggctacctcacttc ctttgagatg tttaacagca cttataagct ctatacacat 2940 aggtgaggacggggacaggg aagaagaata tttmwtkttg tatgatksty ytamctktss 3000 maagcwtkctcaaatctstk aytkyatctg attmgcaaaa acaaagdctg tgccaattcc 3060 ctaaggcctatcaactgaaa cccggwccac ttacaaagcc ggaggagcct aagaggcttc 3120 tccattcttggcctcaaaag cattaatata tgacttaaga gtcaaaagtt ttggstgggg 3180 cagtggcttcatgcctgtaa tccctgcact ttgggaggcc gaggtgggtg ggtcacctga 3240 ggtcaggcgtttragaccag cctggcaaac atggtgaaac cccgtctyta ctaaaataca 3300 aaaattagctggatatgaca gcgcacacct gtaatcctag ctattcagga ggctgaggca 3360 ggagaatcatttgaaccctg gaggcggaga ttgcagtgag ccgagatcnc accmctgcac 3420 ttcagccggagcgacagagc aagactcagt ctcaaaaaaa aaaaaaaaaa gaatcaaaag 3480 ctttctgtagggagaggaca cttcaagaag gctcaggcaa agctccttgc cagctccttt 3540 gagctggccttcagaggttc agaatccagc ctggaatgtg atcccagttg gggctaggag 3600 ctaagctaaagagagctttt ctgggaatgg ttcctagwgt gggaccctag gaattgtcac 3660 tgtctctggcctttgaatga taactgtggg gaattcttac tgcatagcct tgatccaaac 3720 tgtgcagaaattaccccttg ttgaccacag gagatgaata tgtcacagac agaacaaggt 3780 tttcatctttccagagggac acaggaacaa tgttactttt gaaagaggta gctttaggct 3840 agagaacttcaggaccagca tgaaattagt caatcctgta ttttacagtt acctgggatt 3900 tggattgaaagctgcaagac tagcaaccct gggagccctg gagacagaag gtttgtctgg 3960 gtacctgtgctgggggggga tggtgagggt gacacagata ctccgcttgc ttcttccctt 4020 ccttgatagccattctatgg aggaaaagat tatgttgaat tgggaggcaa atgttgtata 4080 atggacctaataatggcaaa ctccttttct agtttataag ttcagaagtt ttgatgtata 4140 ttattagccatttttagaat gaggtctact tgttcagggg taacagccta tgtctaggca 4200 gctgaagtgtctgcagaaat cccaggcttt acgaatacat tcagcaggag cttgctcaag 4260 ccctgagctttacattggag gcacaggaag cagagtctgt tctacatgca ggtggaacaa 4320 cagagtaactccattgatct cttcacaggt caggcagaac tgggttcagt cccagtgttg 4380 tgatatgaggcragtaacct atctgtgccc ctttcctcac attaaatgag aatttgcatt 4440 taaggcactttgtacagtaa tctgttattg ggatgacatc tattttgcat ttcagagtat 4500 acaaaacatcttcaagtata tttaattgaa gcctctcagc aaccagtgag gaaggtagca 4560 tagcatttctttcctgtttt tataaagggg aaagttgctg takgaaggtt ykrgatctct 4620 twragatgtgatraaagcca tggacccctc tgacaaaagc acatatgcat gaaaatttgc 4680 ttctggtttcagggggttca ccaaccccac aaagcctatc tttgaaccct gagttaagga 4740 ttcctgtcacaggatgttgt catggaatta atttcatagg attttaaggc ccagccccca 4800 tggtgaytcttttccacctc actggcttct tgcttgcctt cctccctctc tctcacttac 4860 ttacctcttaccttgtgccc tggattcttt cagggactga tgggcacact ttccggagtg 4920 cctgtttaccgagatggttg gaagcagagt ggatctttgg gggtgtgaaa taccagtatg 4980 gtggcaaccaagaaggcaag tgatgttttt tcactggtta aagttacgtt tacaatggaa 5040 gctctggaaaagtcccatgg gaaacttttt ccagaactca agagaagctt atcttgttgc 5100 agggasttattccaaagatc ttggcatgcc tccaaggact aatgtgaagt gacagtgaac 5160 aaagcagctgtcattctgca tcagccaagt gtcatggacc cattagatac ctgcccttag 5220 ccaagtgctgtggtgcacat ctattgtcct agctactcca aaggttgagg caagaggatc 5280 acttgagcccatgagttcaa ggctatagtg cgcaatgcca ctgcactcca gcctgggcaa 5340 cagggagaccctacctctta caaattaatt aagaagcata ttctaagcct aggtctaatg 5400 cagcagtgtgaaagcctgtt tagttaatgg ttagctattt aaattatagt aaaacttaaa 5460 accaagacaagaatgattca tcttcttata aaaggtatat acctgaatat caaggaatga 5520 acctgaattcccagtgaagg aagcaggcga gccctttagc tacttgctta caaatgctat 5580 ggaatgtaatgctaggcagc agcacaaggt tggccatgat ctggtgaata cagattaggc 5640 aggagagcggccatggagaa acagactggt gaggctgcag acgtttgctc atctttgttt 5700 tgacgcctcttgtcccaagc ctcagccttc tcctgctttc ttgaccttcc tgctgttccc 5760 tcattgtctccagcagcctg cctcagagag tgtccccttc ccccagcgtc gttctcacct 5820 tacccctgtgcacctttgcc tggcagggga ggtgggcttt gagccctgct atgccgaagt 5880 gctgagggtggtacgaggaa aacttcacca gccagaggag gtccagagag gttccttcta 5940 tgctttctcttactattatg accgagctgt tgacacagac atgattggtg agttcacccc 6000 aggtgtcagtccagagagga aggtggatag ggctgtggtg gggaaggtca aggagaaaga 6060 gcacttgaggtgctttgtcg gggtgattac ccacctcttt tctagtcact cgaacaaaag 6120 ggtggaaatgacttagagtc ttttggaggt gagagatgac caaaacaact atatgaggtc 6180 tttttttttttaacatgttt attgaggtat aattggcata caataagtgc cacatttaaa 6240 gtatacaatttaagttttgt catgtataca cccatgaatc catccagcac attgaagata 6300 ataaacatatttcaccacaa aaagtttcct cctgtctctt tataactttt cttcttatca 6360 caaaagcagtgtttttgcct aactgtgaaa gtatatgtac ctgatctgtc atggcctgag 6420 agagatgaattaatttccta ttattgtggg ggttttgttg ttgttgttgt tttggttttt 6480 tgtttgtttgtttgtttttt gagacagagt ctcactctgt tacccaggct ggagtgcaat 6540 ggcatgatctaggctcactg caacctctgc ctcccgggtt caaccgattc tcctgcccca 6600 gtctcctgagtagctgggat tacaggtgcc tgccaccaca cccggctaat ttttttttta 6660 atagagacgaggtttcacca tgttggtcag gctggtcttg aactcctgac ctcgttatct 6720 gccttcctcggcctcccaaa gtgctgggat tacaggcatg agccaccaca cccggcctat 6780 tgtgttttatgggtctgttt tttccattgt ggttaaatat acataacatg gaatagattg 6840 taaataagtaaattaggttg catagattac attatgtaca tgtgtatata atgaatgaat 6900 gaatgaatttccttatgctt ccttgaaggc gttttgatat cagataatct tctgttttat 6960 ttcagattatgaaaaggggg gtattttaaa agttgaagat tttgaaagaa aagccaggga 7020 agtgtgtgataacttggaaa acttcacctc aggcagtcct ttcctgtgca tggatctcag 7080 ctacatcacagccctgttaa aggatggctt tggctttgca gacagcacag tcttacaggt 7140 aagagacaggacaccagagt ctcataacag ccctcttttg tgggggttga gaaggagtaa 7200 gagcttgttcagtaatcaga gtagctagaa gtgaaattat gaggtatttt tgtttgggct 7260 atggacaaggtactgtgctg ggcaccatga atgtgggaaa ttatctcaat gcaatggtag 7320 cctccgagtgtattaccagg caagctatcg cacaggtcac agaacagaaa gactagcagc 7380 ccaaattaagatgccaagtc acatggttta tttatttatt tatttattta ttattatttt 7440 tttgagacggagtctygctc ttgttkccyr ggctggagtg cartggcryg atcwcrgctc 7500 actgcarcctycrcctcctg ggttcaagcg attctyctgc ctcagcctcc cragtagctg 7560 ggattacaggcrygcgccac cacgccyggc taattttttt gtatttttag tagagacggg 7620 gtttcaccatgttggccagg ctrktctyra actyctgayc tcaggtgatc cacccrcctc 7680 rgcctcccaaagtgctrgra ttayaggyrt gagccaccac kccyrgcctt ttttgktcgk 7740 ttctttttttttchtttttt tttttttttt gagacagggt cttgctctgt cacccatgct 7800 ggagtgcagtggcatgatct cagttcactg caacctctgc ctcccgggtt caagtgaccc 7860 tcccacctcagccctctgag tagctgggat tacaggtgtg tgccaccact cttgtctaat 7920 ttttttgtagagacggggtt ttgccatgtt gcccaggctg gtcttgaact cctggcctca 7980 agcaatccacctgccttggc ctcccaaagt gccaggagta caggcatgag ccactgcgcc 8040 tggccccatgtttggttatt attagtgctt aggaagaggc acttgcttac atagtaggag 8100 ttgagaagcttggtttgttc tttcctaccc ctagatctat tctcacctcc tgaccatgct 8160 ctttctgccacatctattat cattacaagt tgccttatct gaaattagtg aatcagaaaa 8220 taaagcaggggatactttgt gtagtttcaa cgttagggaa agttcagaat actgtctgtc 8280 taaactatctctctagaagg cctgatgggc cacaacctgg gccagaagca ttcagttcag 8340 atatgagaatggtgggtgta ggggcaatgg ccaatgggcc atggccggaa ggaaattgtt 8400 acagagtagtgggaagcctg caaagactgg cttctgtccg ttttgccttg gtttgcccat 8460 gtggatattctttgccaata ttttctgccc aagagctgtg cttgctagag ttggaaactg 8520 gatgaaaaggtgaagacttt ttttcttctc aacagctcac aaagaaagtg aacaacatag 8580 agacgggctgggccttgggg gccacctttc acctgttgca gtctctgggc atctcccatt 8640 gaggccacgtacttccttgg agacctgcat ttgccaacac ctttttaagg ggaggagaga 8700 gcacttagtttctgaactag tctggggaca tcctggactt gagcctagag atttaggttt 8760 aattaattttacacatctaa tagtgaactg ctgcctaacc actcaagagt acacagctgg 8820 caccagagcatcacagagag ccctgtgagc caaaaagtat agttttggaa cttaaccttg 8880 gagtgagagcccagggacag gtccctggaa accaaagaaa aatcgcattt caaccctttg 8940 agtgcctcattccactgaat atttaaattt tcctcttaaa tgggaaactg acttattgca 9000 atcccaagacccatcaatat cagtattttt ttcctcccta tacagggccc tgcccaccct 9060 tatctgcacccacctcccct gaaaaagaga gaaaaaaaaa aamccbggtt ttgctttccw 9120 tgtwtaatycamcgacmcaa aakgggacca tgtcaaaatc tgtwtgatcc tattytgggt 9180 tascyccaatcagccagctg aragccttcc taanttttaw taggatgara gagtaccycc 9240 taactgtgcataaattcagc cttaaaaaaa aaggcacccg ggctttgggg acatgtttgg 9300 gangggggggntgcctcata tacccacctt tggtttaata acattttatc agcactttgg 9360 gataa 9365<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 9 gctacctcac ttcctttgag20 <210> SEQ ID NO 10 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 10 ctggctggtg aagttttcctc 21 <210> SEQ ID NO 11 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 11 gcaggtctcc aaggaagtacg 21 <210> SEQ ID NO 12 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: primer <400> SEQUENCE: 12 gtgagtgctc cctgcatctaacataattcc 30 <210> SEQ ID NO 13 <211> LENGTH: 45 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 13 gatgcagggagcactcacac tagtattcat gtttacacct ttgtg 45 <210> SEQ ID NO 14 <211>LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 14 gcgtagtcct gctgttgccc ctaggtacac tggggtctttttcc 44 <210> SEQ ID NO 15 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 15 gcaacagcaggactacgctt actgccagaa c 31 <210> SEQ ID NO 16 <211> LENGTH: 48 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:16 cccaagcgaa tatgccttcg tcttgtccag tcatgatgct aacactgc 48 <210> SEQ IDNO 17 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 17 cgaaggcata ttcgcttgggttactgtg 28 <210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 18 cttccttcactgggaattca gg 22 <210> SEQ ID NO 19 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:19 ctgtttaccg agatggttgg aagc 24 <210> SEQ ID NO 20 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: primer <400>SEQUENCE: 20 ttaaagcttg ggaaaagaat ggccacttc 29 <210> SEQ ID NO 21 <211>LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 21 agactcgagg tggctcaatg ggagatgcc 29 <210> SEQID NO 22 <211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 22 gcgctgtctc ccacagaggatcgcatcacc atcaccatca caaccagcag acttggtt 58 <210> SEQ ID NO 23 <211>LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 23 aaccaagtct gctggttgtg atggtgatgg tgatgcgatcctctgtggga gacagcgc 58 <210> SEQ ID NO 24 <211> LENGTH: 1601 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 gcgggctgccgcgcaagggt ggcgcgcgcg cgttttcctt gttcctggtc aacaaagaaa 60 tgtggagtgtcttggctgaa tcctcataca gacaagatca ttatggtgct gttaggttga 120 aaaagtgatataataaagga accaaggaga aaattcagaa ggaaagaaaa aattgcctct 180 gcaggtgtgcgagcaggatt gcttctgcaa caaaagcctc cacccagcca catcttggga 240 aaagaatggccacttcttgg ggcacagtct ttttcatgct ggtggtatcc tgtgtttgca 300 gcgctgtctcccacaggaac cagcagactt ggtttgaggg tatcttcctg tcttccatgt 360 gccccatcaatgtcagcgcc agcaccttgt atggaattat gtttgatgca gggagcactg 420 gaactcgaattcatgtttac acctttgtgc agaaaatgcc aggacagctt ccaattctag 480 aaggggaagtttttgattct gtgaagccag gactttctgc ttttgtagat caacctaagc 540 agggtgctgagaccgttcaa gggctcttag aggtggccaa agactcaatc ccccgaagtc 600 actggaaaaagaccccagtg gtcctaaagg caacagcagg actacgctta ctgccagaac 660 acaaagccaaggctctgctc tttgaggtaa aggagatctt caggaagtca cctttcctgg 720 taccaaagggcagtgttagc atcatggatg gatccgacga aggcatatta gcttgggtta 780 ctgtgaattttctgacaggt cagctgcatg gccacagaca ggagactgtg gggaccttgg 840 acctagggggagcctccacc caaatcacgt tcctgcccca gtttgagaaa actctggaac 900 aaactcctaggggctacctc acttcctttg agatgtttaa cagcacttat aagctctata 960 cacatagttacctgggattt ggattgaaag ctgcaagact agcaaccctg ggagccctgg 1020 agacagaagggactgatggg cacactttcc ggagtgcctg tttaccgaga tggttggaag 1080 cagagtggatctttgggggt gtgaaatacc agtatggtgg caaccaagaa ggggaggtgg 1140 gctttgagccctgctatgcc gaagtgctga gggtggtacg aggaaaactt caccagccag 1200 aggaggtccagagaggttcc ttctatgctt tctcttacta ttatgaccga gctgttgaca 1260 cagacatgattgattatgaa aaggggggta ttttaaaagt tgaagatttt gaaagaaaag 1320 ccagggaagtgtgtgataac ttggaaaact tcacctcagg cagtcctttc ctgtgcatgg 1380 atctcagctacatcacagcc ctgttaaagg atggctttgg ctttgcagac agcacagtct 1440 tacaggctgccgtactgagg tgatgggcca agctggagat atccccaaag cccatgttga 1500 caccctgtcctgcaagcgga tggactctgt gggctgcatc cctaagaata aagcagagtt 1560 caggtgtgacctctggcagc aaaaaaaaaa aaaaaaaaaa a 1601 <210> SEQ ID NO 25 <211> LENGTH:405 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 25 MetAla Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 7580 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 9095 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys AlaLeu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe LeuVal Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp GluGly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln LeuHis Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly GlyAla Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr LeuGlu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe AsnSer Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe GlyLeu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr GluGly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg TrpLeu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr GlyGly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala GluVal Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr IleThr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr ValLeu Gln 385 390 395 400 Ala Ala Val Leu Arg 405 <210> SEQ ID NO 26 <211>LENGTH: 2762 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (148)..(1599) <400> SEQUENCE: 26gtggggtcgt atcccgcggg tggaggccgg ggtggcgccg gccggggcgg gggagcccaa 60aagaccggct gccgcctgct ccccggaaaa gggcactcgt ctccgtgggt gtggcggagc 120gcgcggtgca tggaatgggc tatgtga atg aaa aaa ggt atc cgt tat gaa act 174Met Lys Lys Gly Ile Arg Tyr Glu Thr 1 5 tcc aga aaa acg agc tac att tttcag cag ccg cag cac ggt cct tgg 222 Ser Arg Lys Thr Ser Tyr Ile Phe GlnGln Pro Gln His Gly Pro Trp 10 15 20 25 caa aca agg atg aga aaa ata tccaac cac ggg agc ctg cgg gtg gcg 270 Gln Thr Arg Met Arg Lys Ile Ser AsnHis Gly Ser Leu Arg Val Ala 30 35 40 aag gtg gca tac ccc ctg ggg ctg tgtgtg ggc gtg ttc atc tat gtt 318 Lys Val Ala Tyr Pro Leu Gly Leu Cys ValGly Val Phe Ile Tyr Val 45 50 55 gcc tac atc aag tgg cac cgg gcc acc gccacc cag gcc ttc ttc agc 366 Ala Tyr Ile Lys Trp His Arg Ala Thr Ala ThrGln Ala Phe Phe Ser 60 65 70 atc acc agg gca gcc ccg ggg gcc cgg tgg ggtcag cag gcc cac agc 414 Ile Thr Arg Ala Ala Pro Gly Ala Arg Trp Gly GlnGln Ala His Ser 75 80 85 ccc ctg ggg aca gct gca gac ggg cac gag gtc ttctac ggg atc atg 462 Pro Leu Gly Thr Ala Ala Asp Gly His Glu Val Phe TyrGly Ile Met 90 95 100 105 ttt gat gca gga agc act ggc acc cga gta cacgtc ttc cag ttc acc 510 Phe Asp Ala Gly Ser Thr Gly Thr Arg Val His ValPhe Gln Phe Thr 110 115 120 cgg ccc ccc aga gaa act ccc acg tta acc cacgaa acc ttc aaa gca 558 Arg Pro Pro Arg Glu Thr Pro Thr Leu Thr His GluThr Phe Lys Ala 125 130 135 gtg aag cca ggt ctt tct gcc tat gct gat gatgtt gaa aag agc gct 606 Val Lys Pro Gly Leu Ser Ala Tyr Ala Asp Asp ValGlu Lys Ser Ala 140 145 150 cag gga atc cgg gaa cta ctg gat gtt gct aaacag gac att ccg ttc 654 Gln Gly Ile Arg Glu Leu Leu Asp Val Ala Lys GlnAsp Ile Pro Phe 155 160 165 gac ttc tgg aag gcc acc cct ctg gtc ctc aaggcc aca gct ggc tta 702 Asp Phe Trp Lys Ala Thr Pro Leu Val Leu Lys AlaThr Ala Gly Leu 170 175 180 185 cgc ctg tta cct gga gaa aag gcc cag aagtta ctg cag aag gtg aaa 750 Arg Leu Leu Pro Gly Glu Lys Ala Gln Lys LeuLeu Gln Lys Val Lys 190 195 200 gaa gta ttt aaa gca tcg cct ttc ctt gtaggg gat gac tgt gtt tcc 798 Glu Val Phe Lys Ala Ser Pro Phe Leu Val GlyAsp Asp Cys Val Ser 205 210 215 atc atg aac gga aca gat gaa ggc gtt tcggcg tgg atc acc atc aac 846 Ile Met Asn Gly Thr Asp Glu Gly Val Ser AlaTrp Ile Thr Ile Asn 220 225 230 ttc ctg aca ggc agc ttg aaa act cca ggaggg agc agc gtg ggc atg 894 Phe Leu Thr Gly Ser Leu Lys Thr Pro Gly GlySer Ser Val Gly Met 235 240 245 ctg gac ttg ggc gga gga tcc act cag atcgcc ttc ctg cca cgc gtg 942 Leu Asp Leu Gly Gly Gly Ser Thr Gln Ile AlaPhe Leu Pro Arg Val 250 255 260 265 gag ggc acc ctg cag gcc tcc cca cccggc tac ctg acg gca ctg cgg 990 Glu Gly Thr Leu Gln Ala Ser Pro Pro GlyTyr Leu Thr Ala Leu Arg 270 275 280 atg ttt aac agg acc tac aag ctc tattcc tac agc tac ctc ggg ctc 1038 Met Phe Asn Arg Thr Tyr Lys Leu Tyr SerTyr Ser Tyr Leu Gly Leu 285 290 295 ggg ctg atg tcg gca cgc ctg gcg atcctg ggc ggc gtg gag ggg cag 1086 Gly Leu Met Ser Ala Arg Leu Ala Ile LeuGly Gly Val Glu Gly Gln 300 305 310 cct gct aag gat gga aag gag ttg gtcagc cct tgc ttg tct ccc agt 1134 Pro Ala Lys Asp Gly Lys Glu Leu Val SerPro Cys Leu Ser Pro Ser 315 320 325 ttc aaa gga gag tgg gaa cac gca gaagtc acg tac agg gtt tca ggg 1182 Phe Lys Gly Glu Trp Glu His Ala Glu ValThr Tyr Arg Val Ser Gly 330 335 340 345 cag aaa gca gcg gca agc ctg cacgag ctg tgt gct gcc aga gtg tca 1230 Gln Lys Ala Ala Ala Ser Leu His GluLeu Cys Ala Ala Arg Val Ser 350 355 360 gag gtc ctt caa aac aga gtg cacagg acg gag gaa gtg aag cat gtg 1278 Glu Val Leu Gln Asn Arg Val His ArgThr Glu Glu Val Lys His Val 365 370 375 gac ttc tat gct ttc tcc tac tattac gac ctt gca gct ggt gtg ggc 1326 Asp Phe Tyr Ala Phe Ser Tyr Tyr TyrAsp Leu Ala Ala Gly Val Gly 380 385 390 ctc ata gat gcg gag aag gga ggcagc ctg gtg gtg ggg gac ttc gag 1374 Leu Ile Asp Ala Glu Lys Gly Gly SerLeu Val Val Gly Asp Phe Glu 395 400 405 atc gca gcc aag tac gtg tgt cggacc ctg gag aca cag ccg cag agc 1422 Ile Ala Ala Lys Tyr Val Cys Arg ThrLeu Glu Thr Gln Pro Gln Ser 410 415 420 425 agc ccc ttc tca tgc atg gacctc acc tac gtc agc ctg cta ctc cag 1470 Ser Pro Phe Ser Cys Met Asp LeuThr Tyr Val Ser Leu Leu Leu Gln 430 435 440 gag ttc ggc ttt ccc agg agcaaa gtg ctg aag ctc act cgg aaa att 1518 Glu Phe Gly Phe Pro Arg Ser LysVal Leu Lys Leu Thr Arg Lys Ile 445 450 455 gac aat gtt gag acc agc tgggct ctg ggg gcc att ttt cat tac atc 1566 Asp Asn Val Glu Thr Ser Trp AlaLeu Gly Ala Ile Phe His Tyr Ile 460 465 470 gac tcc ctg aac aga cag aagagt cca gcc tca tagtggccga gccatccctg 1619 Asp Ser Leu Asn Arg Gln LysSer Pro Ala Ser 475 480 tccccgtcag cagtgtctgt gtgtctgcat aaaccctcctgtcctggacg tgacttcatc 1679 ctgaggagcc acagcacagg ccgtgctggc actttctgcacactggctct gggacttgca 1739 gaaggcctgg tgctgccctg gcatcagcct cttccagtcacatctggcca gagggctgtc 1799 tggacctggg ccctgctcaa tgccacctgt ctgcctgggctccaagtggg caggaccagg 1859 acagaaccac aggcacacac tgagggggca gtgtggctccctgcctgtcc catccccatg 1919 ccccgtccgc ggggctgtgg ctgctgctgt gcatgtccctgcgatgggag tcttgtctcc 1979 cagcctgtca gtttcctccc cagggcagag ctccccttcctgcaagagtc tgggaggcgg 2039 tgcaggctgt cctggctgct ctggggaagc cgagggacagccataacacc cccgggacag 2099 taggtctggg cggcaccact gggaactctg gacttgagtgtgtttgctct tccttgggta 2159 tgaatgtgtg agttcaccca gaggcctgct ctcctcacacattgtgtggt ttggggttaa 2219 tgatggaggg agacacctct tcatagacgg caggtgcccacctttcaggg agtctcccag 2279 catgggcgga tgccgggcat gagctgctgt aaactatttgtggctgtgct gcttgagtga 2339 cgtctctgtc gtgtgggtgc caagtgcttg tgtagaaactgtgttctgag cccccttttc 2399 tggacaccaa ctgtgtcctg tgaatgtatc gctactgtgagctgttcccg cctagccagg 2459 gccatgtctt aggtgcagct gtgccacggg tcagctgagccacagtccca gaaccaagct 2519 ctcggtgtct cgggccacca tccgcccacc tcgggctgaccccacctcct ccatggacag 2579 tgtgagcccc gggccgtgca tcctgctcag tgtggcgtcagtgtcggggc tgagcccctt 2639 gagctgcttc agtgaatgta cagtgcccgg cacgagctgaacctcatgtg ttccactccc 2699 aataaaaggt tgacaggggc ttctccttca aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2759 aaa 2762 <210> SEQ ID NO 27 <211> LENGTH: 484<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Met LysLys Gly Ile Arg Tyr Glu Thr Ser Arg Lys Thr Ser Tyr Ile 1 5 10 15 PheGln Gln Pro Gln His Gly Pro Trp Gln Thr Arg Met Arg Lys Ile 20 25 30 SerAsn His Gly Ser Leu Arg Val Ala Lys Val Ala Tyr Pro Leu Gly 35 40 45 LeuCys Val Gly Val Phe Ile Tyr Val Ala Tyr Ile Lys Trp His Arg 50 55 60 AlaThr Ala Thr Gln Ala Phe Phe Ser Ile Thr Arg Ala Ala Pro Gly 65 70 75 80Ala Arg Trp Gly Gln Gln Ala His Ser Pro Leu Gly Thr Ala Ala Asp 85 90 95Gly His Glu Val Phe Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly 100 105110 Thr Arg Val His Val Phe Gln Phe Thr Arg Pro Pro Arg Glu Thr Pro 115120 125 Thr Leu Thr His Glu Thr Phe Lys Ala Val Lys Pro Gly Leu Ser Ala130 135 140 Tyr Ala Asp Asp Val Glu Lys Ser Ala Gln Gly Ile Arg Glu LeuLeu 145 150 155 160 Asp Val Ala Lys Gln Asp Ile Pro Phe Asp Phe Trp LysAla Thr Pro 165 170 175 Leu Val Leu Lys Ala Thr Ala Gly Leu Arg Leu LeuPro Gly Glu Lys 180 185 190 Ala Gln Lys Leu Leu Gln Lys Val Lys Glu ValPhe Lys Ala Ser Pro 195 200 205 Phe Leu Val Gly Asp Asp Cys Val Ser IleMet Asn Gly Thr Asp Glu 210 215 220 Gly Val Ser Ala Trp Ile Thr Ile AsnPhe Leu Thr Gly Ser Leu Lys 225 230 235 240 Thr Pro Gly Gly Ser Ser ValGly Met Leu Asp Leu Gly Gly Gly Ser 245 250 255 Thr Gln Ile Ala Phe LeuPro Arg Val Glu Gly Thr Leu Gln Ala Ser 260 265 270 Pro Pro Gly Tyr LeuThr Ala Leu Arg Met Phe Asn Arg Thr Tyr Lys 275 280 285 Leu Tyr Ser TyrSer Tyr Leu Gly Leu Gly Leu Met Ser Ala Arg Leu 290 295 300 Ala Ile LeuGly Gly Val Glu Gly Gln Pro Ala Lys Asp Gly Lys Glu 305 310 315 320 LeuVal Ser Pro Cys Leu Ser Pro Ser Phe Lys Gly Glu Trp Glu His 325 330 335Ala Glu Val Thr Tyr Arg Val Ser Gly Gln Lys Ala Ala Ala Ser Leu 340 345350 His Glu Leu Cys Ala Ala Arg Val Ser Glu Val Leu Gln Asn Arg Val 355360 365 His Arg Thr Glu Glu Val Lys His Val Asp Phe Tyr Ala Phe Ser Tyr370 375 380 Tyr Tyr Asp Leu Ala Ala Gly Val Gly Leu Ile Asp Ala Glu LysGly 385 390 395 400 Gly Ser Leu Val Val Gly Asp Phe Glu Ile Ala Ala LysTyr Val Cys 405 410 415 Arg Thr Leu Glu Thr Gln Pro Gln Ser Ser Pro PheSer Cys Met Asp 420 425 430 Leu Thr Tyr Val Ser Leu Leu Leu Gln Glu PheGly Phe Pro Arg Ser 435 440 445 Lys Val Leu Lys Leu Thr Arg Lys Ile AspAsn Val Glu Thr Ser Trp 450 455 460 Ala Leu Gly Ala Ile Phe His Tyr IleAsp Ser Leu Asn Arg Gln Lys 465 470 475 480 Ser Pro Ala Ser <210> SEQ IDNO 28 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: primer <400> SEQUENCE: 28 cgtatcccgc gggtggaggccggggtg 27 <210> SEQ ID NO 29 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: primer <400> SEQUENCE: 29 cttctgcaagtcccagagcc agtgtgc 27 <210> SEQ ID NO 30 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:30 ggagcccaaa agaccggctg c 21 <210> SEQ ID NO 31 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:31 tgaagtcacg tccaggacag g 21 <210> SEQ ID NO 32 <211> LENGTH: 36 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: primer <400> SEQUENCE:32 cggaattcaa catgaaaaaa ggtaatccgt tatgaa 36 <210> SEQ ID NO 33 <211>LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer <400> SEQUENCE: 33 tgtctagatg aggctggact cttctg 26 <210> SEQ IDNO 34 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: oligonucleotide primer <400> SEQUENCE: 34atcctggact tgagcctaga g 21 <210> SEQ ID NO 35 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: oligonucleotide primer<400> SEQUENCE: 35 ctgatattga tgggtcttgg g 21 <210> SEQ ID NO 36 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:oligonucleotide primer <400> SEQUENCE: 36 ggatggaaag gagttggtca g 21<210> SEQ ID NO 37 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: oligonucleotide primer <400> SEQUENCE: 37gtccacatgc ttcacttcct c 21 <210> SEQ ID NO 38 <211> LENGTH: 502 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 38 Met Glu AspThr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile LeuAla Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu AlaVal Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr GlyIle Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr LysTrp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80 ValGlu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 LysVal Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu GluGly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys PheSer Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr AsnAsn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser ThrGln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro AspAsn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn ValTyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu TrpGln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile LeuArg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn ValSer Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met ThrLeu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn TyrGln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330 335 TyrCys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys PheSer Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His PheThr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile GlnGly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu ThrAsn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser HisSer Thr Tyr Val Phe Leu 465 470 475 480 Met Val Leu Phe Ser Leu Val LeuPhe Thr Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile Phe His Lys Pro 500<210> SEQ ID NO 39 <211> LENGTH: 465 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 39 Met Ala Thr Ser Trp Gly Ala Val Phe Met LeuIle Ile Ala Cys Val 1 5 10 15 Gly Ser Thr Val Phe Tyr Arg Glu Gln GlnThr Trp Phe Glu Gly Val 20 25 30 Phe Leu Ser Ser Met Cys Pro Ile Asn ValSer Ala Gly Thr Phe Tyr 35 40 45 Gly Ile Met Phe Asp Ala Gly Ser Thr GlyThr Arg Ile His Val Tyr 50 55 60 Thr Phe Val Gln Lys Thr Ala Gly Gln LeuPro Phe Leu Glu Gly Glu 65 70 75 80 Ile Phe Asp Ser Val Lys Pro Gly LeuSer Ala Phe Val Asp Gln Pro 85 90 95 Lys Gln Gly Ala Glu Thr Val Gln GluLeu Leu Glu Val Ala Lys Asp 100 105 110 Ser Ile Pro Arg Ser His Trp GluArg Thr Pro Val Val Leu Lys Ala 115 120 125 Thr Ala Gly Leu Arg Leu LeuPro Glu Gln Lys Ala Gln Ala Leu Leu 130 135 140 Leu Glu Val Glu Glu IlePhe Lys Asn Ser Pro Phe Leu Val Pro Asp 145 150 155 160 Gly Ser Val SerIle Met Asp Gly Ser Tyr Glu Gly Ile Leu Ala Trp 165 170 175 Val Thr ValAsn Phe Leu Thr Gly Gln Leu His Gly Arg Gly Gln Glu 180 185 190 Thr ValGly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr Phe 195 200 205 LeuPro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr Leu 210 215 220Thr Ser Phe Glu Met Phe Asn Ser Thr Phe Lys Leu Tyr Thr His Ser 225 230235 240 Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly Ala245 250 255 Leu Glu Ala Lys Gly Thr Asp Gly His Thr Phe Arg Ser Ala CysLeu 260 265 270 Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val LysTyr Gln 275 280 285 Tyr Gly Gly Asn Gln Glu Gly Glu Met Gly Phe Glu ProCys Tyr Ala 290 295 300 Glu Val Leu Arg Val Val Gln Gly Lys Leu His GlnPro Glu Glu Val 305 310 315 320 Arg Gly Ser Ala Phe Tyr Ala Phe Ser TyrTyr Tyr Asp Arg Ala Ala 325 330 335 Asp Thr His Leu Ile Asp Tyr Glu LysGly Gly Val Leu Lys Val Glu 340 345 350 Asp Phe Glu Arg Lys Ala Arg GluVal Cys Asp Asn Leu Gly Ser Phe 355 360 365 Ser Ser Gly Ser Pro Phe LeuCys Met Asp Leu Thr Tyr Ile Thr Ala 370 375 380 Leu Leu Lys Asp Gly LeuGly Phe Ala Glu Arg His Pro Leu Thr Ala 385 390 395 400 His Lys Glu SerGlu Gln His Arg Asp Trp Leu Gly Leu Gly Gly His 405 410 415 Leu Ser ProAla Pro Val Ser Gly His His Gln Leu Arg Pro Ser Ser 420 425 430 Thr SerGlu Ala Cys Ile Ser Glu Pro Val Phe Ser Gln Glu Gly Val 435 440 445 AspSer Glu Thr Phe Ser Asp Leu Ser Gly Lys Ala Trp Pro Glu Thr 450 455 460Arg 465

What is claimed is:
 1. A method of hydrolyzing nucleotide diphosphatemolecules at a higher rate than nucleotide triphosphate moleculescomprising the step of contacting a medium comprising nucleotidediphosphates with an effective amount of a nucleotide diphosphatase(NDPase) selected from the group consisting of a CD39-L4 polypeptidehaving NDPase activity and comprising an amino acid sequence with atleast about 90% sequence identity to SEQ ID NO: 3 and a CD39-L2polypeptide having NDPase activity and comprising an amino acid sequencewith at least about 90% sequence identity to SEQ ID NO:
 27. 2. Themethod of claim 1 wherein the NDPase is a CD39-L4 polypeptide havingNDPase activity and comprising an amino acid sequence with at leastabout 90% sequence identity to SEQ ID NO:
 3. 3. The method of claim 2wherein the CD39-L4 polypeptide comprises the amino acid sequence of SEQID NO: 3 or the mature protein portion thereof.
 4. The method of claim 1wherein the NDPase is a CD39-L2 polypeptide having NDPase activity andcomprising an amino acid sequence with at least about 90% sequenceidentity to SEQ ID NO:
 27. 5. The method of claim 4 wherein the CD39-L2polypeptide comprises the amino acid sequence of SEQ ID NO: 27 or themature protein portion thereof.
 6. The method of claim 1 wherein thenucleotide diphosphate molecules comprise adenosine diphosphates (ADPs).7. The method of claim 1 wherein the nucleotide diphosphate moleculescomprise cytidine diphosphates (CDPs).
 8. The method of claim 1 whereinthe nucleotide diphosphate molecules comprise guanosine diphosphates(GDPS).
 9. The method of claim 1 wherein the nucleotide diphosphatemolecules comprise thymidine diphosphates (TDPs).
 10. The method ofclaim 1 wherein the nucleotide diphosphate molecules comprise uridinediphosphates (UDPs).
 11. The method of claim 1 wherein said nucleotidediphosphates are hydrolyzed in a mammalian subject.
 12. A method ofreducing the ratio of adenosine nucleoside diphosphates (ADPs) toadenosine triphosphates (ATPs) (ADP:ATP) in a mammalian subjectcomprising the step of administering to said mammalian subject aneffective amount of a nucleotide diphosphatase (NDPase) selected fromthe group consisting of a CD39-L4 polypeptide having NDPase activity andcomprising an amino acid sequence with at least about 90% sequenceidentity to SEQ ID NO: 3 and a CD39-L2 polypeptide having NDPaseactivity and comprising an amino acid sequence with at least about 90%sequence identity to SEQ ID NO:
 27. 13. The method of claim 12 whereinsaid ratio is reduced by administration of CD39-L4 having the sequenceset forth in SEQ ID NO: 3 or a polypeptide having NDPase activity and atleast about 90% sequence identity to SEQ ID NO:
 3. 14. The method ofclaim 12 wherein said ratio is reduced by administration of CD39-L2having the sequence set forth in SEQ ID NO: 27 or a polypeptide havingNDPase activity and at least about 90% sequence identity to SEQ ID NO:27.
 15. The method of claim 12 wherein the ratio of ADP:ATP is reducedsystemically in circulation.
 16. The method of claim 12 wherein theratio of ADP:ATP is reduced locally within an area selected from thegroup consisting of heart, brain, kidney, lung and limbs.
 17. The methodof claim 12 wherein the ratio of ADP:ATP is reduced withoutsignificantly affecting ATP levels.